Escherichia coli

Nucleic Acids Res, 1991 Nov 25, 19(22), 6295 - 300
Effect of RecF protein on reactions catalyzed by RecA protein; Madiraju MV et al.; RecF protein is one of at least three single strand DNA (ssDNA) binding proteins which act in recombination and repair in Escherichia coli . In this paper we show that our RecF protein preparation complexes with ssDNA so as to retard its electrophoretic movement in an agarose gel . The apparent stoichiometry of RecF-ssDNA-binding measured in this way is one RecF molecule for every 15 nucleotides and the binding appears to be cooperative . Interaction of the other two ssDNA-binding proteins, RecA and Ssb proteins, has been studied extensively; so in this paper we begin the study of the interaction of RecF and RecA proteins . We found that the RecF protein preparation inhibits the activity of RecA protein in the formation of joint molecules whether added before or after addition of RecA protein to ssDNA . It, therefore, differs from Ssb protein which stimulates joint molecule formation when added to ssDNA after RecA protein . We found that our RecF protein preparation inhibits two steps prior to joint molecule formation: RecA protein binding to ssDNA and coaggregate formation between ssDNA-RecA complexes and dsDNA . We found that it required a much higher ratio of RecF to RecA protein than normally occurs in vivo to inhibit joint molecule formation . The insight that these data give to the normal functioning of RecF protein is discussed.

J Biol Chem, 1991 Nov 25, 266(33), 22386 - 91
Nitrate-inducible formate dehydrogenase in Escherichia coli K-12 . II . Evidence that a mRNA stem-loop structure is essential for decoding opal (UGA) as selenocysteine; Berg BL et al.; fdnG, encoding the selenopeptide of Escherichia coli formate dehydrogenase-N, contains an in-frame opal (UGA) codon at amino acid position 196 that directs selenocysteine incorporation . We have identified sequences that contribute to the mRNA context required for decoding this UGA as selenocysteine . We identified a potential stem-loop structure immediately downstream of UGA196 that is comparable in size and structure to a stem-loop predicted to form in fdhF, which encodes the selenopeptide of E . coli formate dehydrogenase-H . Mutational analysis of the fdnG stem-loop structure suggests that it is critical for decoding UGA196 as selenocysteine . Our data indicate that both stability and specific nucleotide sequences of the stem-loop likely contribute to the appropriate mRNA context for selenocysteine incorporation into the fdnG gene product.

J Biol Chem, 1991 Nov 25, 266(33), 22380 - 5
Nitrate-inducible formate dehydrogenase in Escherichia coli K-12 . I . Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine; Berg BL et al.; The fdnGHI operon of Escherichia coli encodes nitrate-inducible formate dehydrogenase . We report here the entire nucleotide sequence of fdnGHI . The sequence contains three open reading frames of sizes appropriate to encode the three subunits of formate dehydrogenase-N . fdnG contains an in-frame UGA codon that specifies selenocysteine incorporation, and the predicted amino acid sequence of FdnG shows similarity to two other bacterial formate dehydrogenase enzymes . FdnH contains 4 cysteine clusters typical of those found in iron-sulfur proteins . FdnG also contains a cysteine cluster . Evidence from sequence and spectral analyses suggest that FdnI encodes cytochrome bFdn556 . Implications for the membrane topology of formate dehydrogenase-N and its mechanism of proton translocation are discussed.

J Biol Chem, 1991 Nov 25, 266(33), 22141 - 6
One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters; Moriyama Y et al.; About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes . The recombinant plasmid pBWU13 carries the unc operon for F0F1 . When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential . Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis . F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.

J Theor Biol, 1991 Nov 21, 153(2), 255 - 85
Control theory of regulatory cascades; Kahn D et al.; We have extended Metabolic Control Theory to include cascades consisting of several modules controlling each other solely via regulatory effects . We derive several theorems that determine how the control properties of a cascade derive from (1) the control properties of each module, taken in isolation and (2) the regulatory interactions between the modules . Two cases are treated explicitly . The first concerns cascades in the absence of feed-back: in this case the internal control behaviour of each module is unaffected by external regulatory interactions . The second includes one feed-back loop and gives a quantitative expression of how feed-back modifies control properties: the internal control matrix within one module can be calculated as if the elasticity matrix of this module was the sum of its intrinsic elasticity matrix and a cyclic regulation matrix . More complex cascades can be analysed recursively by subdividing them into simpler modules, which can be treated individually . The theoretical framework developed here should facilitate quantitative experimental analysis of the control of cell physiology where the latter involves regulatory cascades.

J Mol Biol, 1991 Nov 20, 222(2), 373 - 90
Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor; van Mierlo CP et al.; An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser . Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein . The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable . Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures . The folded and unfolded conformations are in slow exchange . The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.

J Mol Biol, 1991 Nov 20, 222(2), 353 - 71
(14-38, 30-51) double-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor: a two-dimensional 1H nuclear magnetic resonance study; van Mierlo CP et al.; An analogue of the BPTI folding intermediate that contains only the disulphide bonds between Cys14 and Cys38 and between Cys30 and Cys51 has been prepared in Escherichia coli by protein engineering methods . The other two Cys residues of native BPTI (at positions 5 and 55) have been replaced by Ser . Essentially complete proton resonance assignments of the analogue were obtained by employing two-dimensional 1H nuclear magnetic resonance techniques . The intermediate has a more extended conformation in the N-terminal (residues 1 to 7) region and there are other differences in the C-terminal (residues 55 to 58) region . The remainder of the protein is substantially identical to native BPTI . The conformational properties of the analogue can explain several aspects of the kinetic role that the normal (14-38, 30-51) intermediate plays in the folding of BPTI.

J Mol Biol, 1991 Nov 20, 222(2), 265 - 80
Absolute in vivo translation rates of individual codons in Escherichia coli . The two glutamic acid codons GAA and GAG are translated with a threefold difference in rate; Sorensen MA et al.; We have determined the absolute translation rates for four individual codons in Escherichia coli . We used our previously described system for direct measurements of in vivo translation rates using small, in-frame inserts in the lacZ gene . The inserts consisted of multiple synthetic 30 base-pair DNA oligomers with high densities of the four individual codons, GAA (Glu), GAG (Glu), CCG (Pro) and CGA (Arg) . Our method is independent of expression level, of mRNA half-life and of transcription rate . Codon GAA was found to be translated with a rate of 21.6 codons/second whereas codon GAG was translated 3.4-fold slower (6.4 codons/s) . These two codons are read by the same tRNA species . Codon CCG and CGA are both read by abundant tRNA species but nevertheless we found them to be translated slowly with rates of 5.8 and 4.2 codons/second, respectively . The context of these codons were varied, but we found no significant influence of context on their translation rates and we suggest a mechanism for why context may not affect translation rates . One insert with a low translation rate gave results that most readily can be explained by assuming queue formation of ribosomes on the insert . Such a queue was found to reduce the expression level by approximately 35% . Our experiments allowed us to estimate the average distance between ribosomes and thereby the translation initiation frequency on the wild-type lacZ mRNA . This was found to be one per three seconds.

J Mol Biol, 1991 Nov 20, 222(2), 251 - 64
Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli; Egebjerg J et al.; The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities . The results show that the sites are confined to localized RNA regions within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier . They are as follows: (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions . (2) The L2 site constitutes a single irregular stem/loop structure in the centre of domain IV where non-Watson-Crick pairing is likely to occur . (3) L23 recognizes a tertiary structural motif involving a single terminal loop structure and part of an adjacent internal loop at the centre of domain III . Each of the three primary binding proteins, whose presence is essential for ribosomal assembly, has been associated with important ribosomal functions: L1 lies in the E-site for deacylated tRNA binding while L2 and L23 have been implicated in the P and A substrate sites, respectively, of the peptidyl transferase centre . Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region.

J Mol Biol, 1991 Nov 20, 222(2), 139 - 42
Expression of the protease inhibitor ecotin and its co-crystallization with trypsin; McGrath ME et al.; We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter . After induction, functional, soluble ecotin comprises 15% of total cellular protein . This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin . Ecotin was co-crystallized with rat trypsin mutant D102N . Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21 . The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees . Four molecules each of trypsin and ecotin are found in the asymmetric unit.

J Mol Biol, 1991 Nov 20, 222(2), 189 - 96
An Escherichia coli rpoB mutation that inhibits transcription of catabolite-sensitive operons; Rockwell P et al.; The Escherichia coli rpoB636 mutant is defective in the transcription of lac and other catabolite-sensitive operons . The lac promoter variant, UV5, which is independent of cyclic AMP and the cyclic AMP receptor protein, CRP, was also defective in rpoB636 mutants . The activity of the lac UV5 promoter was restored to wild-type levels by deletion of cya (adenylate cyclase) or crp . Cyclic AMP and CRP apparently act as inhibitors of the rpoB636 RNA polymerase.

Biochemistry, 1991 Nov 19, 30(46), 11092 - 103
Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis; Warren MS et al.; We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H) . The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution . The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain . A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22 . Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining . Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range . For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values . While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered . Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme . We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.

Biochemistry, 1991 Nov 19, 30(46), 11073 - 80
Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme; Boles JO et al.; Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques {Yang et al . (1990) Science (Washington, D.C.) 249, 1398-1405} . The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet . We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E . coli Rue10 . An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E . coli in 90% yield in no more than 8 h . The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values . Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h) . The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen . Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes . In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.

Biochemistry, 1991 Nov 19, 30(46), 11046 - 54
The function of amino acid residues contacting the nicotinamide ring of NADPH in dihydrofolate reductase from Escherichia coli; Adams JA et al.; The importance of three amino acid residues contacting the nicotinamide ring of NADPH in Escherichia coli dihydrofolate reductase has been defined using site-directed mutagenesis and detailed steady-state and pre-steady-state kinetic experiments . Replacement of Tyr-100 with either glycine or isoleucine (Y100G or Y100I) disrupts an aromatic-aromatic interaction between the phenolic side chain and the nicotinamide ring . Both mutations remove the differential binding of the oxidized and reduced coenzymes implicating Tyr-100 as a major determinant for coenzyme specificity . Replacement of Ser-49 for alanine (S49A), designed to either displace or reduce the polarizability of a bound water molecule contacting the N1 of the nicotinamide ring, affects only the rate of release of NADP+ . Replacement of Ile-14 with alanine (I14A), designed to alter both a weakly polar and a hydrogen bonding interaction with the periphery of the nicotinamide ring, affects only the binding of NADPH . Y100I, Y100G, and I14A all increase the activation barrier for the chemical step by approximately 2 kcal/mol . The lack of an effect for S49A suggests that water structure is not important for stabilizing the hydride transfer transition state . In addition, the nominal effects observed for these mutations disfavor the hypothesis that neighboring amino acid residues participate in the stabilization of the reaction transition state through polar or weakly polar contacts.

Biochemistry, 1991 Nov 19, 30(46), 11054 - 63
Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II; Fierke CA et al.; Twelve amino acid substitutions of varying size and hydrophobicity were constructed at Val 143 in human carbonic anhydrase II (including Gly, Ser, Cys, Asn, Asp, Leu, Ile, His, Phe and Tyr) to examine the catalytic roles of the hydrophobic pocket in the active site of this enzyme . The CO2 hydrase and p-nitrophenyl acetate (PNPA) esterase activities, the pKa of the zinc-water ligand, the inhibition constant for cyanate (KOCN), and the binding constants for sulfonamide inhibitors were measured for various mutants and correlated with the size and hydrophobicity of the substituted amino acid . The kcat/KM for PNPA hydrolysis and KOCN are linearly dependent on the hydrophobicity of the amino acid at position 143 . All of the activities of CAII are decreased by more than a factor of 10(3) when large amino acids (Phe and Tyr) are substituted for Val 143, but the CO2 hydrase activity is the most sensitive to the size and structure of the substituted amino acid . Addition of a single methyl group (V143I) decreases the activity 8-fold, while substitution of valine by tyrosine essentially destroys the enzyme function (kcat/KM for CO2 hydration is decreased by more than 10(5)-fold) . KOCN does not increase until Phe is substituted for Val 143, suggesting that the cyanate and CO2 binding sites are not identical . The functional data in conjunction with X-ray crystallographic studies of four of the mutants {Alexander et al., 1991 (following paper in this issue)} allow interpretation of the mutants at a molecular level and mapping of the region of the active site important for CO2 association . The hydrophobic pocket, including residues Val 121 and Val 143, is important for CO2 and PNPA association; if the pocket is blocked, substrates cannot approach the zinc-hydroxide with the correct orientation to react . The interaction between Val 143 and CO2 is relatively weak (less than or equal to 0.5 kcal/mol) and nonspecific; the association site does not tightly hold CO2 in one fixed orientation for reaction with the zinc-hydroxide . This mechanism of catalysis may reflect a decreased requirement for specific orientation by CO2 since it is a symmetrical molecule.

FEBS Lett, 1991 Nov 18, 293(1-2), 21 - 4
P26-calcium binding protein from bovine retinal photoreceptor cells; Kutuzov MA et al.; The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA . This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina . Preliminary data are presented on expression of P26 as a fusion protein in E . coli.

FEBS Lett, 1991 Nov 18, 293(1-2), 164 - 8
Bacterioferritins and ferritins are distantly related in evolution . Conservation of ferroxidase-centre residues; Andrews SC et al.; Iron-storage proteins can be divided into two classes; the bacterioferritins and ferritins . In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously . This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by 'Profile Analysis' . Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins.

FEBS Lett, 1991 Nov 18, 293(1-2), 160 - 3
Impaired affinity for phenylalanine in Escherichia coli phenylalanyl-tRNA synthetase mutant caused by Gly-to-Asp exchange in motif 2 of class II tRNA synthetases; Kast P et al.; Phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 subunit structure) is a member of class II of tRNA synthetases . We report here the genetic analysis of an Escherichia coli mutant strain which is auxotrophic for phenylalanine because it has a PheRS with a decreased affinity for phenylalanine . The mutant pheS gene encoding the PheRS alpha subunit was cloned and sequenced, and the deviation from the wild-type gene was found to result in a Gly191-to-Asp191 exchange . This alteration is located within motif 2, one of 3 conserved sequence motifs characteristic for class II aminoacyl-tRNA synthetases . Motif 2 may thus participate in the formation of the phenylalanine binding site in PheRS.

FEBS Lett, 1991 Nov 18, 293(1-2), 106 - 10
Purification and characterization of human T-cell leukemia virus type I protease produced in Escherichia coli; Kobayashi M et al.; Human T-cell leukemia virus type I (HTLV-I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli . The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease . Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease . The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24 . The protease activity was inhibited by pepstatin A . These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV-I virion.

FEBS Lett, 1991 Nov 18, 293(1-2), 34 - 6
Mutational analysis of the putative PLA2-inhibiting sequence of annexin 1; Trave G et al.; Annexin 1 has been proposed to inhibit phospholipase A2 by direct interaction through a specific amino acid sequence spanning residues 246-254 . The possible role of this region was investigated by protein engineering . Three point mutations and a deletion have been performed . The four mutant proteins have been expressed in E . coli, purified and tested for calcium and lipid binding, and for phospholipase inhibition . All mutant proteins conserved the properties of the wild-type recombinant protein . This result clearly demonstrates that this part of the molecule is not involve in the inhibition of phospholipase A2.

Tijdschr Diergeneeskd, 1991 Nov 15, 116(22), 1122 - 9
{Colibacillosis in poultry: etiology, pathology and therapy}; Goren E; Colibacillosis is a secondary infection caused by inhalation, particularly in young broilers and poults . The severity of this septicaemic disease is due to a combination of factors as the virulence, exposure to aerogenic infection, the E . coli strains involved and the intensity of the predisposing alteration of the respiratory epithelium by viruses . Mycoplasmas and/or CO2, NH3 and dust in the atmosphere . Despite preventive vaccination against several predisposing viral infections and the successful eradication of M . gallisepticum in the Netherlands, colibacillosis continues to be an important disease in the broiler industry . Treatment is confronted with a number of serious problems: high costs when treating flocks with the correct (relatively high) dosage for a sufficiently long period, resistance to bacterial drugs and drug residues . Despite negligible resorption from the gastrointestinal tract, very satisfactory therapeutic results were obtained by oral treatment, even against in vitro resistant E . colli strains . To explain the mode of action it is suggested that well-absorbed metabolites or degradation products of spectinomycin block the adhesion of bacteria to tissue receptors.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 9909 - 13
Mutant profiles of selectable genetic elements; Wurst H et al.; A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function . It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide . A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose . The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window . Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide . When this procedure was performed for both DNA strands, all mismatches could be detected . A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo . This method, called "mutant profiling," should be applicable to all selectable genetic elements.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10193 - 7
Replication of acetylaminofluorene-adducted plasmids in human cells: spectrum of base substitutions and evidence of excision repair; Mah MC et al.; In rats fed the liver carcinogen 2-acetylaminofluorene (AAF), the two most abundant types of DNA adduct are N-(deoxyguanosin-8-yl)-2-acetylaminofluorene and its deacetylated derivative . When plasmids carrying AAF adducts replicate in bacteria, the predominant mutations are frameshifts, whereas with deacetylated (AF) adducts, they are mainly base substitutions, just as we found when plasmids carrying AF adducts replicated in human cells . We have investigated the frequency and spectrum of mutations induced when a shuttle vector carrying AAF adducts (85% bound to the C8 position of guanine, 15% to the N2 position) replicated in human cells . The frequency induced per initial AAF adduct was higher than with AF adducts, but the kinds of mutations were similar--i.e., 85% base substitutions, principally G.C----T.A transversions . There was good correlation between the "hot spots" for mutations and hot spots for AAF adduct formation, suggesting that mutational hot spots reflect preferential binding of the carcinogen to DNA . 32P-postlabeling analysis of the adducts before and after the DNA was transfected into the human cells showed that there was no deacetylation of AAF adducts and that 85% of both types of adducts were removed within 3.5 hr, most probably by excision repair.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10049 - 53
Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines; Wilkie TM et al.; Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) . These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site . G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages . Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells . Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA . The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function.

J Biol Chem, 1991 Nov 15, 266(32), 22057 - 62
Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase; Nguyen T et al.; The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein . The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems . After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein . In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala . Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time . High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus . The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants . These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.

J Biol Chem, 1991 Nov 15, 266(32), 21657 - 65
Mechanism-based inactivation of alanine racemase by 3-halovinylglycines; Thornberry NA et al.; Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3-chloro- and 3-fluorovinylglycine . Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 +/- 0.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine . The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7 . The reversible adduct (lambda max = 516 nm) decays to regenerate free enzyme with a half-life of 23 min . The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg . The second-order rate constant for this process with D-chlorovinylglycine (122 +/- 14 M-1 s-1), the most reactive analog examined, is faster than the equivalent rate constant for D-fluoroalanine (93 M-1 s-1) . The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chemistry.

J Biol Chem, 1991 Nov 15, 266(32), 21608 - 15
Functional characteristics of the rrnD promoters of Escherichia coli; Langert W et al.; The function of the tandem rrnD promoters (P1, P2) of Escherichia coli, which are highly efficient in directing rRNA synthesis, was studied in vitro using the strong hybrid promoter PtacI as a reference . One of the characteristics of the rrnD promoters is a pronounced instability of binary and initiating complexes formed with RNA polymerase . The rate of productive complex formation and of chain initiation at these promoters was found to be limited by a step in binary complex transitions with an apparent first-order rate constant equal to 3.9 x 10(-2) s-1 . A comparison of this rate with that determined previously by filter binding assays (Gourse, R . (1988) Nucleic Acids Res . 16, 9789-9809) suggests that the rate-limiting step is a conversion of an intermediate species of open complex to one that is efficient in productive initiation . The slow rate of this reaction and the instability of open complexes account for the relatively low competitive strengths of the rrnD promoters . However, this limitation of rrn promoter function changes with promoter occupancy because the rate of chain initiation increased after completion of the first round of initiation . Despite their poor competitive strength, the rrnD promoters are more productive than PtacI at nonlimiting RNA polymerase concentrations . This can be ascribed to the different rates with which RNA polymerases leave PtacI and the rrnD promoters . These functional differences of the promoters are consistent with a "stressed intermediate" model of chain initiation (Straney, D.C., and Crothers, D.M . (1987) J . Mol . Biol . 193, 267-278) which predicts that rapid clearance of the rrn promoters is mechanistically related to the instability of the binary complexes.

J Biol Chem, 1991 Nov 15, 266(32), 21458 - 65
Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains; Baksh S et al.; Recombinant calreticulin and discrete domains of calreticulin were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and their Ca2+ binding properties were determined . Native calreticulin bound 1 mol of Ca2+/mol of protein with high affinity, and also bound approximately 20 mol of Ca2+/mol of protein with low affinity . Both Ca2+ binding sites were present in the recombinant calreticulin indicating that proper folding of the protein was achieved using this system . Calreticulin is structurally divided into three distinct domains: the N-domain encompassing the first 200 residues; the P-domain which is enriched in proline residues (residue 187-317); and the C-domain which covers the carboxyl-terminal quarter of the protein (residues 310-401), and contains a high concentration of acidic residues . These domains were expressed in E . coli, isolated, and purified, and their Ca2+ binding properties were analyzed . The C-domain bound approximately 18 mol of Ca2+/mol of protein with a dissociation constant of approximately 2 mM . The P-domain bound approximately 0.6-1 mol of Ca2+/mol of protein with a dissociation constant of approximately 10 microM . The P-domain and the C-domain, when expressed together as the P+C-domain, bound Ca2+ with both high affinity and low affinity, reminiscent of both full length recombinant calreticulin and native calreticulin . In contrast the N-domain, did not bind any detectable amount of 45Ca2+ . We conclude that calreticulin has two quite distinct types of Ca2+ binding sites, and that these sites are in different structural regions of the molecule . The P-domain binds Ca2+ with high affinity and low capacity, whereas the C-domain binds Ca2+ with low affinity and high capacity.

Eur J Biochem, 1991 Nov 15, 202(1), 53 - 66
The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution; Arrowsmith C et al.; We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data . This is the largest protein structure thus far determined by NMR . To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing . Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides . Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization . We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid . An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10 . Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously {C . H . Arrowsmith, R . Pachter, R . B . Altman, S . B . Iyer & O . Jardetzky (1990) Biochemistry 29, 6332-6341} . Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here . The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter . The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS)

Arch Biochem Biophys, 1991 Nov 15, 291(1), 107 - 12
Studies of ligand binding to Escherichia coli adenylosuccinate synthetase; Soans C et al.; Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme . The enzyme has one binding site for each of these ligands . Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined . These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme . Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding . The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands . TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site . Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate . These results suggest that the IMP and aspartate binding sites are spatially separated.

Cell, 1991 Nov 15, 67(4), 807 - 14
Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex; Gray JT et al.; Telomeres of Oxytricha nova macronuclear chromosomes consist of a repeated T4G4 sequence, single-stranded at the 3' terminus, bound by a heterodimeric protein . The cloning of genes for the two polypeptides and their separate expression in E . coli have enabled evaluation of their individual contributions to DNA binding . The 56 kd alpha subunit binds single-stranded DNA by itself, one polypeptide per T4G4 block; multiple subunits can coat a (T4G4)n multimer . The derived amino acid sequence of alpha does not reveal any known DNA-binding motif, so it appears to represent a novel type of DNA-binding protein . The previously cloned 41 kd beta subunit does not by itself protect DNA from methylation, but is required along with alpha to recreate the pattern of methylation protection indicative of telomeres in vivo . The unusual ability of the protein to engage in two different interactions with the same telomeric DNA sequence might provide the versatility necessary for diverse telomere functions.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 9989 - 93
Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation; Webster LC et al.; Trans-activation by the adenovirus E1A 289R protein requires a zinc finger defined by Cys-154, Cys-157, Cys-171, and Cys-174 . Whereas individually replacing the four cysteine residues with serines resulted in a loss of transactivation, only three of the Cys----Ser mutants (C157S, C171S, and C174S) lost the ability to bind Zn(II) . X-ray absorption fine structure analysis revealed that, in the wild-type protein, Zn(II) is coordinated by four cysteine residues whereas in the C154S mutant, Zn(II) is coordinated by two histidines and two cysteines . The mutant protein probably retains, as ligands, two cysteines on the right side of the zinc finger (Cys-171 and Cys-174) and recruits two of the four histidines on the left side (His-149, His-152, His-158, and His-160), despite the presence of Cys-157 . This finding may shed light on the general structural requirements of zinc fingers.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10153 - 7
Relationship between alternatively spliced exons and functional domains in tropomyosin; Cho YJ et al.; Smooth and striated muscle alpha-tropomyosins differ as a consequence of alternative splicing of exons 2 and 9 encoding amino acid residues 39-80 and 258-284, respectively {Ruiz-Opazo, N., Weinberger, J . & Nadal-Ginard, B . (1985) Nature (London) 315, 67-70} . To understand the relationship between alternatively spliced exons and functional domains in tropomyosin, recombinant unacetylated striated muscle, smooth muscle, and chimeric rat alpha-tropomyosins (+H3N-tropomyosins) expressed in and purified from Escherichia coli were analyzed . The functional differences between the isoforms can be primarily ascribed to exon 9 . +H3N-Tropomyosins with the smooth muscle exon 9 bound to skeletal muscle filamentous actin with at least a 5-fold higher affinity than +H3N-tropomyosins with the striated muscle exon 9 . On the other hand, in the presence of Ca2+, troponin increased the affinity of +H3N-tropomyosins with the striated muscle exon 9 at least 50-fold, whereas it had little effect on +H3N-tropomyosins with the smooth muscle exon 9 . The unique striated muscle alpha-tropomyosin exon 9 seems to be specialized for Ca(2+)-insensitive interaction with troponin on the thin filament . The unique smooth muscle alpha-tropomyosin exon 2 was associated with a slightly lower actin affinity than the striated muscle exon 2 . Although the regions encoded by exons 2 and 9 correspond to functional domains, they are not recognizable as independent units or structural domains in the extended coiled-coil structure of this fibrous actin binding protein.

J Biol Chem, 1991 Nov 15, 266(32), 21700 - 8
Early events in the transport of proteins into mitochondria . Import competition by a mitochondrial presequence; Cyr DM et al.; Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G . (1991) J . Biol . Chem . 266, 21693-21699) . Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20 . A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2 . Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import . Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein . Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition . F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25% . In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25% . Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi . Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions . F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step . Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide . We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.

J Biol Chem, 1991 Nov 15, 266(32), 21693 - 9
Interaction of peptides corresponding to mitochondrial presequences with membranes; Hoyt DW et al.; The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus . Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane . To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry . These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure . Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior . Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer . High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors . Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site . These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.

Anal Biochem, 1991 Nov 15, 199(1), 119 - 24
A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-galactosidase in lacZ-transfected cells; Jain VK et al.; An optimized chemiluminescent assay for beta-galactosidase using a chemiluminescent substrate AMPGD (3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo-{3.3.1 . 1(3,7)}decan}-4- yl)phenyl-beta-D-galactopyranoside) is described . This assay is rapid and sensitive and can detect as little as 2 fg of beta-galactosidase . Its use for the quantitation of beta-galactosidase in cells transfected with lacZ-expressing vectors is described . It is possible to detect a single cell stably expressing lacZ by this technique.

Gene, 1991 Nov 15, 107(2), 241 - 6
Three individual regulatory elements of the promoter positively activate the transcription of the murine gene encoding granulocyte colony-stimulating factor; Asano M et al.; At least three regulatory elements GPE1, GPE2 and GPE3 (G-CSF promoter elements) controlling the gene (G-CSF) encoding granulocyte colony-stimulating factor (G-CSF) are indispensable for the constitutive expression of the G-CSF gene in human CHU-2 cells and for its lipopolysaccharide(LPS)-inducible expression in macrophages . The enhancer activities of each regulatory element were examined with or without the SV40 enhancer element placed downstream from the reporter gene . A GPE1 tetramer mediated the constitutive expression in CHU-2 cells, and the LPS-inducible expression in macrophage cell lines, while the GPE2 element was active in CHU-2 and LPS-treated macrophage cell lines only in combination with the SV40 enhancer . A GPE3 tetramer had efficient enhancer activity in CHU-2 cells but not in macrophage cell lines without the SV40 enhancer . In combination with the SV40 enhancer, GPE3 worked as an LPS-inducible enhancer element in macrophage BAM3 cells . Gel retardation assay indicated that the CHU-2 and the macrophage cells contained nuclear factors which specifically bound to each GPE sequence.

Biochem J, 1991 Nov 15, 280 ( Pt 1), 187 - 91
Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119; Fillat MF et al.; The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR) . The gene is transcribed into a 1250-base transcript . The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron . The PCR-amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli . High concentrations of flavodoxin were found (20% of total protein) . The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin.

J Biol Chem, 1991 Nov 15, 266(32), 21777 - 83
Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants; Kumar R et al.; In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D . R., and Stanley, P . (1990) J . Biol . Chem . 265, 1615-1622) . One of these transfectants was used to make a genomic DNA library in lambda ZAP-II from EcoRI-digested, size-selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified . After in vivo excision, two plasmids with DNA inserts greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants . A partial restriction map of this clone revealed an approximately 3.6-kb PstI fragment that contained an Alu sequence . This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(1,3)-Fuc-T gene (Lowe, J . B., Stoolman, L . M., Nair, R . P., Larsen, R . D., Berhend, T . L., and Marks, R . M . (1990) Cell 63, 475-484) . Both approximately 3.6-kb fragments gave identical restriction patterns . In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4){Fuc alpha(1,3)}GlcNAc beta 1 but not the alpha(2,3)-sialyl-Lex determinant . As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex is a carbohydrate ligand recognized by ELAM-1 . Surprisingly, however, an open reading frame encoded within the approximately 3.6-kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S . E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356) . Possible explanations for these apparently disparate results are discussed.

Eur J Biochem, 1991 Nov 15, 202(1), 95 - 9
Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G; Brinkmann T et al.; The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor . Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity . Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine . Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10(-8) M . Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.

FEMS Microbiol Lett, 1991 Nov 15, 68(2), 227 - 30
Amplification by the polymerase chain reaction of a specific target sequence in the gene coding for Escherichia coli verotoxin (VTe variant); Johnson WM et al.; Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to target a specific sequence in the gene coding for the A subunit of Escherichia coli verotoxin (VTe-variant, VTev) . This PCR protocol permits the VTe-variant target sequence to be distinguished from closely related sequences in the same coding regions for type 1, type 2, and type 2 variant E . coli verotoxins . This procedure will be a valuable adjunct to other DNA amplification techniques currently being used for molecular epidemiological studies of verotoxigenic E . coli.

Biochim Biophys Acta, 1991 Nov 15, 1080(3), 259 - 63
Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form; Hammarstrom A et al.; Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest . The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay . There is no difference in pH optimum between the forms in the biosynthetic assay . In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+) . It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay . The two forms of Rsp . rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors . In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition . In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive . The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation . It is also shown that the low-activity form of Rsp . rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.

FEMS Microbiol Lett, 1991 Nov 15, 68(2), 135 - 8
Isolation and preliminary characterization of the Porphyromonas gingivalis prtC gene expressing collagenase activity; Takahashi N et al.; A gene, prtC, has been isolated from Porphyromonas gingivalis ATCC 53977 in Escherichia coli utilizing the plasmid vector pPL-lambda . The resultant protease positive clone NHS1, harboring plasmid pS1 with a 5.9-kilobase P . gingivalis insert, expressed an enzyme capable of hydrolyzing the synthetic collagenase substrate PZ-PLGPA as well as solubilized type I collagen . Subcloning and deletion analysis located the prtC gene at one end of the P . gingivalis DNA insert on plasmid pS1.

Gene, 1991 Nov 15, 107(2), 307 - 12
The hygromycin-resistance-encoding gene as a selection marker for vaccinia virus recombinants; Zhou J et al.; Hygromycin B (Hy), an inhibitor of RNA translation, was shown to block the replication of vaccinia virus (VV) in cultured cell lines . Insertion of the Escherichia coli Hy resistance-encoding gene (hph) into the VV genome under control of early or late synthetic VV promoters could overcome inhibition of viral replication . When hph was inserted into VV in tandem with the human papillomavirus type 16 (HPV16) L1 open reading frame, hph recombinant viruses could be selected which expressed HPV16 L1.

Biochem J, 1991 Nov 15, 280 ( Pt 1), 233 - 41
The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc . Evidence for the involvement of a zinc-dependent structure in phorbol ester binding; Ahmed S et al.; Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes . DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid . Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC . Until recently, PKC was the only known phorbol ester receptor . We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement {Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem . J . 272, 767-773} . The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14) . The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions) . The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions . We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E . coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding . The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound {3H}phorbol 12,13-dibutyrate . When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited . These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.

Biochem J, 1991 Nov 15, 280 ( Pt 1), 125 - 9
Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase; Takazawa K et al.; In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein {clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem . J . 272, 107-112} . Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes . The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way . The catalytic domain is thus located at the C-terminal end of the protein . A series of 5' deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids . Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain . Affinity chromatography on CaM-Sepharose of 5' and 3' deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation.

Biochim Biophys Acta, 1991 Nov 15, 1080(3), 271 - 4
Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus . Orientation of porin in the membrane; Welte W et al.; By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced . With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates . Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE . According to this result a single channel of approx . 30 A diameter starts at the outer surface . Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10297 - 301
Cloning and characterization of a 20-kDa ubiquitin carrier protein from wheat that catalyzes multiubiquitin chain formation in vitro; Van Nocker S et al.; Recent evidence indicates that the commitment to degrade cellular proteins by the ubiquitin proteolytic pathway is dependent on the covalent attachment of multiubiquitin chains to the target protein {Chau, V., Tobias, J . W., Bachmair, A., Marriott, D., Ecker, D . J., Gonda, D . K . & Varshavsky, A . (1989) Science 243, 1576-1583} . We have isolated a 20-kDa ubiquitin carrier protein {E2(20 kDa)} from wheat by using ubiquitin covalent affinity chromatography and anion-exchange HPLC that catalyzes multiubiquitin chain formation in vitro . This reaction is blocked by the addition of a mutant ubiquitin in which arginine has been substituted for lysine at residue 48, demonstrating that the coupling of ubiquitin to ubiquitin is likely to be through an isopeptide linkage between the C-terminal glycine and Lys48 of ubiquitin . By immunoscreening a wheat cDNA expression library with anti-E2(20 kDa) antibodies, a cDNA encoding the complete protein was isolated . The clone (designated UBC7) was confirmed as encoding E2(20 kDa) by comparison of the derived amino acid sequence with peptide sequences of E2(20 kDa) tryptic fragments . The encoded protein contains a single cysteine at position 91, which is presumably the active site, and has regions of amino acid sequence similarity to other known E2s from plants and yeast . Expression of this cDNA in Escherichia coli produced an active E2 capable of catalyzing multiubiquitin chain formation in vitro . By virtue of its activity, E2(20 kDa) may have a pivotal role in protein degradation by the ubiquitin-dependent proteolytic pathway.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10168 - 72
Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413; Mannan RM et al.; In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two {4Fe-4S} centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex . An insertion mutation introduced in the psaC locus of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide . In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark . However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased . In intact cells of T398-1, bicarbonate-dependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates . The low-temperature EPR signals attributed to the {4Fe-4S} centers FA and FB were absent in the mutant cells . Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells . Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI . Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB . These studies demonstrate that Anabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.

J Biol Chem, 1991 Nov 15, 266(32), 21753 - 9
Expression of a sodium proton antiporter (NhaA) in Escherichia coli is induced by Na+ and Li+ ions; Karpel R et al.; Regulation of expression of nhaA, the gene which encodes a Na+/H+ antiporter in Escherichia coli has been studied . Two promoters have been identified in the upstream sequence of the gene and the corresponding start point of transcription mapped by primer extension . Monitoring the beta-galactosidase activity of a chromosomal translation fusion of nhaA'-'lacZ show that at pH 7.5 the gene is induced, within 1 h, by 100 mM of either Li+ or Na+ . Change of pH between 6.5 and 8.5 by itself does not increase expression of the gene but it markedly increases the sensitivity of the expression system to the ions . At pH 7.5 maximal induction is obtained by 100 mM NaCl, whereas at pH 8.6, 10 mM NaCl elicit similar response . The pattern of regulation of nhaA reflects its importance in adaptation to high salinity and alkaline pH in E . coli.

J Biol Chem, 1991 Nov 15, 266(32), 21736 - 44
Purification of TnsB, a transposition protein that binds to the ends of Tn7; Arciszewska LK et al.; We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7 . The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein . The apparent molecular mass of denatured TnsB protein is approximately 85 kDa . Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape . Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon . The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition . Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7 . These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.

J Biol Chem, 1991 Nov 15, 266(32), 21404 - 8
Interferon-inducible mouse Mx1 protein that confers resistance to influenza virus is GTPase; Nakayama M et al.; The murine Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection even though the resistance mechanism is yet unclear . The Mx1 protein contains a tripartite GTP-binding domain consisting of GXXXXGKS, DXXG, and T/NKXD motifs . In the GTPase gene superfamily such as p21ras protein, signal-transducing G protein, and translation elongation factor, the GTPase activity plays a key role in each protein function . Here we show that GTPase activity is indeed associated with the intact Mx1 protein purified from Escherichia coli expressing Mx1 cDNA . Amino acid substitution within the GTP-binding motif led to significant reduction in the GTPase activity . Yeast vacuolar protein sorting (VPS1) protein and the rat microtubule-associated mechanochemical enzyme dynamin were found to be homologous to Mx1 not only in the tripartite GTP-binding motif, but also in the amino-terminal region of approximately 300 amino acids in length . The function of Mx1 is discussed in comparison with these proteins.

Biochim Biophys Acta, 1991 Nov 14, 1115(1), 49 - 53
Radiofrequency dielectric spectroscopy of ribosome suspensions; Bonincontro A et al.; Dielectric measurements on different ribosome suspensions were carried out in the frequency range from 10 kHz to 1 GHz . In intact ribosomes two dispersions were detected: one around 100 kHz and the other one in the MHz region . In separated ribosomal subunits and in ribosomes resuspended in a buffer with no magnesium ions (relaxed ribosomes) only the MHz dispersion was observed . Electrical conductivities of the samples at 1 kHz were also measured . The temperature dependence of the two dispersions was investigated and a tentative attribution was proposed.

Biochim Biophys Acta, 1991 Nov 14, 1115(1), 36 - 41
Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase; Shioda T et al.; To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined . E . coli harboring the vector plasmid was used as a control . Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG) . TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG . TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG . Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains . These results indicate that chicken AK1 expressed in E . coli catalyzed the synthesis and accumulation of TTP within the bacterial cells.

Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1408 - 15
Engineering by PCR-based exon amplification of the genomic porcine interferon-gamma DNA for expression in Escherichia coli; Vandenbroeck K et al.; Interferon-gamma (IFN-gamma) is coded for by a single gene containing three introns, localized within the coding region . We have previously cloned the IFN-gamma gene from a pig genomic DNA lambda library and have determined its nucleotide sequence . In order to construct the porcine IFN-gamma DNA without intervening sequence, the four exons were separately amplified by the polymerase chain reaction (PCR) using primers matching the exon-termini . From the amplified exon-fragments the complete intron-free DNA was obtained by a strategy consisting of alternate rounds of PCR and ligation . The sequence so-obtained was used for expression in E . coli . The recombinant protein appeared as inclusion bodies which were solubilized and refolded in order to obtain biologically active IFN-gamma.

Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1222 - 6
ClpB proteins copurify with the anaerobic Escherichia coli reductase; Pontis E et al.; Two proteins, called alpha and beta 3, copurify with the anaerobic ribonucleotide reductase from Escherichia coli (Eliasson et al . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 3314-3318) . Both are now identified as products of the clpB gene that is presumed to code for a subunit of an ATP dependent protease . The tight associations suggest the possibility that the ClpB proteins are involved in the regulation of the anaerobic reductase.

Nature, 1991 Nov 14, 354(6349), 161 - 4
FtsZ ring structure associated with division in Escherichia coli; Bi EF et al.; Genes for cell division have been identified in Escherichia coli by the isolation of conditional lethal mutations that block cell division, but do not affect DNA replication or segregation . Of these genes, ftsZ is of great interest as it acts earliest in the division pathway, is essential, its level dictates the frequency of division, and it is thought to be the target of two cell-division inhibitors, SulA, produced in response to DNA damage, and MinCD, which prevents division at old sites . Here we have used immunoelectronmicroscopy to localize the FtsZ protein to the division site . The results suggest that FtsZ self-assembles into a ring structure at the future division site and may function as a cytoskeletal element . The formation of this ring may be the point at which division is regulated.

Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1476 - 82
Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin; Sugimoto M et al.; We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase . Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit . The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites . However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit . This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.

Biochemistry, 1991 Nov 12, 30(45), 10920 - 4
RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay; Tyagarajan K et al.; We have used a self-cleaving RNA molecule (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase . Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3'-deoxynucleoside triphosphates . When the nascent transcript attains the minimum length required for the "hammerhead" domain of the transcript to fully emerge from the ternary complex, the "hammerhead" structure forms and self-cleaves, producing a truncated product . The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to that generated by using a noncleaving control template . We have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site . The results indicate that the RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization . The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10 . The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed.

Biochemistry, 1991 Nov 12, 30(45), 10914 - 20
An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21; Manor D et al.; We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family . In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site . A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange . We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems . The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding . On the other hand, no change in hydrogen bonding is observed at guanine's N7 . The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond . These results are compared to previous structural and chemical studies.

Biochemistry, 1991 Nov 12, 30(45), 10872 - 7
NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain; Lowry DF et al.; The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods . Five proton resonances were found below 10.5 ppm . Two of these were assigned to the amide groups of Lys 24 and Gly 83 . These are conserved residues in each of the consensus sequences . Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 {Redfield, A . G., & Papastavros, M . Z . (1990) Biochemistry 29, 3509-3514} . The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21 . These results suggest that this conserved lysine has a similar structural role in proteins in this class . The tentative Gly 83 resonance has no spectral analogue in p21 . A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.

Biochemistry, 1991 Nov 12, 30(45), 10832 - 8
Selecting high-affinity binding proteins by monovalent phage display; Lowman HB et al.; Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system . Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor . The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle . After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor . Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method . However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated . This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.

Biochemistry, 1991 Nov 12, 30(45), 10987 - 91
Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase; Mendel-Hartvig J et al.; The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies . The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site . Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo . The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-{3-(dimethylamino)propyl}-carbodiimide (EDC) . In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced . Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding . When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+ . When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Nov 12, 30(45), 10844 - 9
Gene structure and expression of the rat cytochrome P450IIC13, a polymorphic, male-specific cytochrome in the P450IIC subfamily; Eguchi H et al.; The male-specific CYP2C13 gene has been isolated from two independent rat genomic libraries . This gene spans more than 50 kb and contains eight introns which are subject to the GT-AG rule . Two allelic forms of the CYP2C13 gene were identified . Determination of the exonic sequences revealed that one of them encodes cytochrome P450(+g) and the other encodes cytochrome P450(-g) . Using allele-specific restriction enzyme sites, a good correlation between the genotype and the phenotype of CYP2C13 was shown . Nucleotide substitutions between the (+g) and the (-g) genes exist not only in the exons but also in the introns and the 5'-flanking region . Although five nucleotide differences were identified within 287 base pairs of the (+g) and (-g) 5'-flanking regions, the transcription initiation sites were identical . In addition to a canonical TATA box located 31 base pairs upstream of the start site of transcription, putative binding sites for the liver-enriched and liver-specific transcription factors HNF1/LF-B1/APF, HNF3, HNF4/AF-1, C/EBP, LAP, and eH-TF/TGT3 and the ubiquitous factors NF-1 and OTF-1 were identified.

Am J Obstet Gynecol, 1991 Nov, 165(5 Pt 1), 1568 - 74
A rabbit model for bacterially induced preterm pregnancy loss: intervention studies with ampicillin-sulbactam; McDuffie RS Jr et al.; We conducted experiments with a previously described rabbit model of Escherichia coli-induced preterm pregnancy loss . Does at 70% gestation were inoculated hysteroscopically with 0.2 ml of Escherichia coli (10(5) colony-forming units per milliliter) or saline solution . Animals were randomly assigned to either receive treatment with ampicillin-sulbactam (begun 1 to 2 hours before inoculation and continued for up to 7 days) or to receive no therapy . Animals were killed after delivery or after 7 days . Saline solution-inoculated animals had no pregnancy loss . Of the Escherichia coli-inoculated animals, those treated with ampicillin-sulbactam had significantly fewer deliveries, fewer positive cultures, and more live fetuses than the untreated animals (p less than or equal to 0.001) . Cultures from multiple sites, amniotic fluid prostaglandin levels, and maternal progesterone levels were obtained, and the placenta, uterus, and fetal lung were histologically evaluated . In the second phase of the study, the Escherichia coli-inoculated animals were treated with ampicillin-sulbactam at one of three times: at inoculation or 2 or 4 hours after inoculation . The Escherichia coli-inoculated does treated with ampicillin-sulbactam at or before inoculation had significantly fewer deliveries, fewer positive cultures, and more live fetuses than the Escherichia coli-inoculated does in which treatment was delayed 4 hours (p less than or equal to 0.01).

J Bacteriol, 1991 Nov, 173(21), 6991 - 7
Isolation and expression of a gene cluster responsible for biosynthesis of the glycopeptidolipid antigens of Mycobacterium avium; Belisle JT et al.; Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS . Serovars of the M . avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces . A genomic library of DNA from serovar 2 of the M . avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M . avium serovar 2-specific glycopeptidolipid . The responsible gene cluster was mapped to a 22- to 27-kb functional region of the M . avium genome . The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M . smegmatis, whereas the oligosaccharide segment arose from the cloned M . avium genes . This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes.

Res Microbiol, 1991 Nov-Dec, 142(9), 943 - 9
Differences in codon usage among genes encoding proteins of different function in Rhodobacter capsulatus; Wu LF et al.; Codon usage in Rhodobacter was evaluated and found to be strikingly different from that in Escherichia coli . While codon usage for genes concerned with nitrogen utilization and carotenoid biosynthesis corresponded to expectation, based on codon usage for Rhodobacter in general, that for the fructose utilization (fru) operon and for the photosynthetic genes encoding the reaction centre and light harvesting proteins exhibited significant deviation from expectation and from each other for specific amino acids . The differences in codon usage for the fru operon versus the photosynthetic genes may reflect different proportions of the various tRNA specific for certain amino acids when cells are grown under heterotrophic versus phototropic conditions . In addition, preferential use of the initiation codon, GTG, was found for the first cistrons of Rhodobacter operons.

Nucleic Acids Res, 1991 Nov 11, 19(21), 6033 - 40
SQUIRREL: Sequence QUery, Information Retrieval and REporting Library . A program package for analyzing signals in nucleic acid sequences for the VAX; Gartmann CJ et al.; A computer tool is described for comparison, analysis and search of genetic signals . The method is based on sequence consensus matrices . It assumes that a genetic signal (such as a promoter, enhancer or whatever) is composed of several signal blocks separated from each other by variable distances . A set of programs is presented to perform the analysis . The result of such an analysis is a description of the investigated signal including matrices for each signal block, distances between each block and distribution of the values . Programs are provided to search for a signal using results from previous analysis . The method is able to align large sets of sequences within a few minutes and to check the quality of the alignment . An analysis of E.coli promoters is provided as an example.

Nucleic Acids Res, 1991 Nov 11, 19(21), 6007 - 13
Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands; Ciccarelli RB et al.; A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis . Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated . The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer . Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique . Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene . The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis . In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's . Results suggest that sequence errors arose from both DNA synthesis and PCR . The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5839 - 42
W (A or T) sequences as probes and primers suitable for genomic mapping and fingerprinting; Drmanac R et al.; A limitation to the use of oligonucleotide probes as tools for genetic and physical mapping has been the low hybridization positive frequency obtained by oligonucleotides of sufficient length to hybridize preferentially to cloned insert DNA (and not host E . coli genomic DNA) . Both computer and experimental results now indicate that oligonucleotide probes composed of W (A or T) sequence are preferentially found in eukaryotic DNA, and can be used to provide high frequency, discriminative hybridization . Such W sequences may be useful as either probes or PCR primers in molecular diagnostic applications as well as in genetic and physical mapping.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5863 - 70
A novel RNA product of the tyrT operon of Escherichia coli; Bosl M et al.; The tyrT operon of E . coli and several other tRNA operons of E . coli show striking structural features: they contain repeated sequence units including a 19bp motif resembling the 3' end of the corresponding mature tRNA . A novel RNA, encoded by the repeated sequence of the tyrT operon, was identified . The RNA, characterized by primer extension and Nuclease-S1 analysis, contained 171 nucleotides and terminated with the 19bp motif of the CCA-end of tRNA(1Tyr) . The RNA, designated as rtT RNA, is probably released from the primary transcript of tyrT during tRNA processing, it includes the coding capacity for the arginine rich peptide Tpr . Predictions of secondary folding resulted a rather stable RNA structure with a free energy of -44.3kcal/mol . A weak ribosome binding site was found, preceding the second possible AUG initiator codon for Tpr . The comparison of rtT RNA with putative transcripts from the repeated sequences associated with related tRNA genes showed common features with respect to primary structure, arrangement and secondary folding . In E . coli cultures the lag-phase during growth, caused by transient glycine or by isoleucine limitation, was found to be overcome or markedly shortened in the presence of rtT RNA . These and previously reported results suggest a modulatory effect of rtT RNA on stringent response.

Biochim Biophys Acta, 1991 Nov 11, 1090(3), 293 - 8
Differential expression in Escherichia coli of the alpha and beta forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure; Forough R et al.; Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed . When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield . However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector . Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5907 - 14
Two distinct human DNA diesterases that hydrolyze 3'-blocking deoxyribose fragments from oxidized DNA; Chen DS et al.; Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA . We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis . Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells . The two activities were purified to differing degrees from HeLa cells . One enzyme had the properties of the known HeLa AP endonuclease (Mr approximately 38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein . The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity . This second enzyme may have been detected in other studies but never characterized . In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3'-deoxyribose diesterase activities . It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected . The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5889 - 94
Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin; Sedgwick SG et al.; Antibody-binding epitopes in the central helical region of the muscular dystrophy protein, dystrophin, have been mapped using a new strategy of transposon mutagenesis . Tn1000 transposons carrying translation termination codons were introduced randomly by bacterial mating into a large fragment of dystrophin cDNA in a pEX2 plasmid to produce a library of transformants expressing truncated dystrophin fusion proteins . Epitopes were progressively lost as the expressed sequences were shortened, enabling the epitopes recognised by 22 monoclonal antibodies to be placed in order along the dystrophin molecule without in vitro manipulation of DNA . The C-terminus of each truncated fusion protein was precisely located within the dystrophin sequence by direct sequencing of pEX2 transformants using transposon-specific primers . Sequences as short as 7 and 17 amino-acids have been identified as essential for antibody binding in this way . Nineteen of the 22 monoclonal antibodies had been selected for their ability to bind both native and SDS-denatured dystrophin and 15 of these bind to one sequence of 74 amino-acids (residues 1431-1505 of the 3684 residue sequence) . This may be an area of high immunogenicity or of close structural similarity between native dystrophin and the SDS-treated recombinant fragment used for immunization.

Biochim Biophys Acta, 1991 Nov 11, 1090(3), 317 - 25
Binding characteristics of Escherichia coli biotin repressor-operator complex; Lin KC et al.; The genes of the operon bioA.BFCD are transcribed divergently from a single regulatory region between the bioA and, bioB genes . Transcription in both directions is co-repressed by biotinyl-5'-adenylate and the biotin repressor . The multimeric state of the biotin repressor bound to DNA and how it affects transcription have not been fully characterized . Therefore, we isolated the BirA protein from a recombinant strain which overproduced the biotin repressor and studied the repressor-operator binding characteristics through restriction enzyme site protection experiments and the mobility shift assay . We also measured the stoichiometry of the biotin repressor-operator complex directly . The results of restriction enzyme site protection studies are consistent with the postulation that the biotin operator is approx . 40 bp long . Only one retarded DNA band appeared in the mobility shift experiments, suggesting that the repressor-operator binding could be a single step reaction . The repressor-operator binding stoichiometry determination revealed that two repressor monomers may occupy the wild type or half palindromic biotin operator sequences.

Biochem Pharmacol, 1991 Nov 6, 42(11), 2131 - 9
Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213); Mans DR et al.; In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro . The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively . The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation . The radical introduced, in addition, chemical DNA adducts . Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA . The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts {van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retel J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988} . When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced . In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity . It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

J Mol Biol, 1991 Nov 5, 222(1), 99 - 124
Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations; Kast P et al.; Neither the tertiary structure nor the location of active sites are known for phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 structure), a member of class II aminoacyl-tRNA synthetases . In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically . The pheS mutations were found to cause Ala294 to Ser294 exchanges in the alpha subunits from both independent strains . This alteration (S294) resided in the well-conserved C-terminal part of the alpha subunit, precisely within motif 3, a typical class II tRNA synthetase sequence . We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS . Mutation S294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294 . The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids . In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability . However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues . Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe . Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo . The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed.

J Biol Chem, 1991 Nov 5, 266(31), 21174 - 8
Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase; Watanabe T et al.; Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat . Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues . To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells . We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429 . GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant . By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel . Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant) . However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition . Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E . coli alkaline phosphatase) . Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding . The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E . coli alkaline phosphatase), respectively.

J Biol Chem, 1991 Nov 5, 266(31), 21118 - 24
Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins; Shaw JP et al.; Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia . To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF . The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM . Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF . Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine . Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h . Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies . These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.

J Biol Chem, 1991 Nov 5, 266(31), 20946 - 52
Site-directed mutagenesis studies on the recombinant thioesterase domain of chicken fatty acid synthase expressed in Escherichia coli; Pazirandeh M et al.; The animal fatty acid synthase is a multifunctional protein with a subunit molecular weight of 260,000 . We recently reported the expression and characterization of the acyl carrier protein and thioesterase domains of the chicken liver fatty acid synthase in Escherichia coli . In order to gain insight into the mechanism of action of the thioesterase domain, we have replaced the putative active site serine 101 with alanine and cysteine and the conserved histidine 274 with alanine by site-directed mutagenesis . While both the Ser101----Ala and His274----Ala mutant proteins were inactive, the Ser101----Cys mutant enzyme (thiol-thioesterase) retained considerable activity, but the properties of the enzyme were changed from an active serine esterase to an active cysteine esterase, providing strong evidence for the role of Ser101 as the active site nucleophile . In order to further probe into the role of His274, a double mutant was constructed containing both the Ser101----Cys and the His274----Ala mutations . The double-mutant protein was inactive and exhibited diminished reactivity of the Cys-SH to iodoacetamide as compared to that of the Ser101----Cys-thioesterase, suggesting a role of His274 as a general base in withdrawing the proton from the Cys-SH in the thiol-thioesterase or Ser101 in the wild-type enzyme . Incubation of the recombinant thioesterases with {1-14C} palmitoyl-CoA resulted in the incorporation of {1-14C} palmitoyl into the enzyme only in the double mutant, suggesting that Cys-SH of the double mutant is reactive enough to form the palmitoyl-S-enzyme intermediate . This intermediate is not hydrolyzed because of the lack of His274, which is required for the attack of H2O on the acyl enzyme . These results suggest that the catalytic mechanism of the thioesterases may be similar to that of the serine proteases and lipases, which employ a serine-histidine-aspartic acid catalytic triad as part of their catalytic mechanism.

J Biol Chem, 1991 Nov 5, 266(31), 20928 - 33
Characteristics of the gene for a spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome; Furuchi T et al.; The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined . It contained four open reading frames encoding A, B, C, and D proteins . By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity . A single transport system was involved in the transport of both spermidine and putrescine . The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions . In addition, it had consensus amino acid sequences for the nucleotide binding site . B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles . D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein . These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system.

J Biol Chem, 1991 Nov 5, 266(31), 20922 - 7
Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome; Kashiwagi K et al.; The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined . It contained the genes for an induced ornithine decarboxylase and a putrescine transport protein . The gene for the ornithine decarboxylase contained a 2,196-nucleotide open reading frame encoding a 732-amino acid protein whose calculated Mr was 82,414, and the predicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min . The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0 . The gene for the putrescine transport protein contained a 1,317-nucleotide open reading frame encoding a 439-amino acid protein whose calculated Mr was 46,494 . The hydropathy profile of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length . The transport protein was in fact found in the membrane fraction . When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly . The results suggest that the gene expression of the operon is repressed strongly under standard conditions.

J Biol Chem, 1991 Nov 5, 266(31), 20870 - 6
Membrane insertion of the Escherichia coli MalF protein in cells with impaired secretion machinery; McGovern K et al.; The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids . We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes . Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF . In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins . Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes . Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery.

J Biol Chem, 1991 Nov 5, 266(31), 20840 - 8
Identification of the human platelet GTPase activating protein for the CDC42Hs protein; Hart MJ et al.; The CDC42Hs protein appears to be an isoform of the ras-related GTP-binding protein G25K and is an apparent human homolog of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc . In this study, we report the identification of a GTPase-activating protein (GAP) for CDC42Hs from human platelets (designated from here on as CDC42Hs-GAP) . The CDC42Hs-GAP activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being Mono-Q chromatography), and purified approximately 3500-fold . The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent Mr of approximately 25,000 . The GTPase activities of the purified human platelet CDC42Hs, the Escherichia coli-recombinant CDC42Hs, and the Spodoptera frugiperda-recombinant GTP-binding proteins are all stimulated by the CDC42Hs-GAP to identical extents, which indicates that the recombinant CDC42Hs proteins are as effective as the native human platelet protein in coupling to the GAP . However, a mutant form of the E . coli-recombinant CDC42Hs which contains a valine residue at position 12 (CDC42HsVal-12) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42HsGly-12) which is not stimulated by the CDC42Hs-GAP . The CDC42Hs-GAP also does not stimulate the GTPase activities of the ras or rap GTP-binding proteins; however, it is capable of a weak stimulation of the GTPase activity of mammalian rho . Based on the apparent similarities in the molecular size of the CDC42Hs- and rho-GAPs (i.e . 25-30 kDa), and the cross-reactivity of rho with the CDC42Hs-GAP, it seems likely that the CDC42Hs- and rho-GAPs will constitute a specific subclass of the ras-related GAP superfamily.

J Biol Chem, 1991 Nov 5, 266(31), 20781 - 5
Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase . Histidine 142 alpha is a facilitative catalytic residue; Luo GX et al.; There are 11 histidine residues in Escherichia coli succinyl-CoA synthetase . His-246 alpha is well established as the phosphorylation site of the enzyme . Replacement of this histidine by asparagine (Mann, C . J., Mitchell, T., and Nishimura, J . S . (1991) Biochemistry 30, 1497-1503) or by aspartic acid (Majumdar, R., Guest, J . R., and Bridger, W . A . (1991) Biochim . Biophys . Acta 1076, 86-90) through site-directed mutagenesis resulted in complete loss of enzyme activity . Chemical modification experiments suggested a second histidine at the active site (Collier, G . E., and Nishimura, J . S . (1979) J . Biol . Chem . 254, 10925-10930) . In the present study, we have changed His-142 alpha to an asparagine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity . The resulting mutant enzyme is practically devoid of enzyme activity but can be thiophosphorylated with adenosine 5'-O-(thiotriphosphate) and dethiophosphorylated with ADP at rates that are significantly faster than those with wild type enzyme . The observation that phosphorylated mutant enzyme can be dephosphorylated with succinate and with succinate plus desulfo-CoA at rates comparable with those with wild type enzyme suggests that mutant enzyme can bind succinate and CoA . Dethiophosphorylation of the enzyme in the presence of CoA plus succinate proceeds much faster with wild type than with mutant . While there was no significant change in KCoA or Ksuccinate, the turnover number for dethiophosphorylation of the mutant was 10-fold lower . These data are consistent with location of His-142 alpha at the active site and a facilitative role for this residue in catalysis.

J Biol Chem, 1991 Nov 5, 266(31), 20773 - 80
Purification and characterization of an endotoxin-binding protein with protease inhibitory activity from Limulus amebocytes; Minetti CA et al.; Using a lipopolysaccharide affinity column and ion exchange chromatography, a 12-kDa protein has been purified from Limulus amebocytes . In solid phase binding assays, the radiolabeled protein binds specifically to lipopolysaccharide (LPS) with a Kd value on the order of 10(-7) M . A cDNA coding for this protein has been isolated and sequenced . The amino acid sequence deduced from the cDNA indicates that this protein shares no sequence homology with LPS-binding proteins isolated from different species of vertebrates (Schumann, R . R., Leong, S . R., Flaggs, G . W., Gray, P . W., Wright, S . D., Mathison, J . C., Tobias, P . S., and Ulevitch, R . J . (1990) Science 249, 1429-1431) and invertebrates (Aketagawa, J., Miyata, T., Ohtsubo, S., Nakamura, T., Morita, T., Hayashida, H., Miyata, T., Iwanaga, S., Takao, T., and Shimonishi, Y . (1986) J . Biol . Chem . 261, 7357-7365) . The binding to LPS can be displaced by the unlabeled 12-kDa protein, polymyxin B, lipid A, and to a lesser extent by D-glucosamine . In whole cell binding assays, the 12-kDa protein has also been shown to bind to Escherichia coli . Using both {14C}casein and a synthetic substrate, the protein has been shown to inhibit the proteolytic activity of trypsin, with an IC50 of approximately 10(-7) M . In the presence of LPS, the antitryptic acitivity of the Limulus endotoxin-binding protein-protease inhibitor remains unaffected . The protein is a major component of the cytoplasmic proteins (1%) . Immunocytochemical analysis reveals that this protein exists in the secretory granules of the amebocytes where enzymes and substrates for the clotting cascade reside . Based on the unusual dual functional properties, the newly isolated protein was named a "Limulus endotoxin-binding protein-protease inhibitor" (LEBP-PI).

J Biol Chem, 1991 Nov 5, 266(31), 20714 - 9
Initiation of protein synthesis in animal mitochondria . Purification and characterization of translational initiation factor 2; Liao HX et al.; Bovine liver mitochondrial translational initiation factor 2 (IF-2mt) has been purified to near homogeneity . The scheme developed results in a 24,000-fold purification of the factor with about 26% recovery of activity . SDS-polyacrylamide gel electrophoresis indicates that IF-2mt has a subunit molecular mass of 85 kDa . IF-2mt promotes the binding of formyl(f)Met-tRNA to mitochondrial ribosomes but is inactive with the nonformylated derivative . IF-2mt is active on chloroplast 30 S ribosomal subunits, but IF-2chl has no activity in promoting fMet-tRNA binding to animal mitochondrial ribosomes . IF-2mt is sensitive to elevated temperatures and is inactivated by treatment with N-ethylmaleimide . It is partially protected from heat and N-ethylmaleimide inactivation by the presence of either GTP or GDP suggesting that guanine nucleotides may bind to this factor directly . The binding of fMet-tRNA to mitochondrial ribosomes requires the presence of GTP and is inhibited by GDP . DeoxyGTP is very effective in replacing GTP in promoting fMet-tRNA binding to ribosomes and some activity is also observed with ITP . No activity is observed with ATP, CTP, or UTP . Nonhydrolyzable analogs of GTP can promote formation of both 28 S and 55 S initiation complexes indicating that GTP hydrolysis is not required for subunit joining in the animal mitochondrial system.

J Biol Chem, 1991 Nov 5, 266(31), 20709 - 13
The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase . Studies by chemical modification, site-directed mutagenesis, and 19F NMR; Donald LJ et al.; Escherichia coli citrate synthase is strongly and specifically inhibited by NADH, but this inhibition can be prevented by reacting the enzyme with Ellman's reagent . We have now labeled the single reactive cysteine covalently with monobromobimane and isolated and sequenced the bimane-containing cyanogen bromide peptide and identified the cysteine as Cys-206 . Modeling studies suggest that this residue is on the subunit surface, 25-30 A from the active site . Mutation of Cys-206 to serine (C206S), or of Gly-207 to alanine (E207A), weakened NADH binding and inhibition; when these mutations were present together, NADH binding was weaker by 18-fold and inhibition by 250-fold . The mutations also had small effects on substrate binding at the active site . Cys-206 of wild type enzyme and of the mutant E207A was alkylated with 1,1,1-trifluorobromoacetone and the environment of the fluorine nuclei studied by 19F NMR . With wild type enzyme, the NMR spectrum consisted of two peaks of about equal intensity but different line widths, at -8.65 ppm (line width 11.2 +/- 0.5 Hz) and -7.6 ppm (line width 57 +/- 4 Hz) . As the labeled wild type citrate synthase was titrated with KCl, the narrow peak converted to the broad one . The same range of KCl concentrations was needed for this conversion as for the allosteric activation of E . coli citrate synthase . The E207A mutant gave the broader NMR peak almost exclusively . We propose that the fluorine label in wild type citrate synthase exists in two conformational states with different mobilities, exchanging slowly on the NMR time scale, and that treatment with KCl, or truncation of the Glu-207 side chain by mutagenesis, stabilizes one of these states . Consistent with this explanation is the finding that Cys-206 reacts more quickly with Ellman's reagent in the presence of KCl, and that this rate is faster yet in the E207A mutant.

Biochemistry, 1991 Nov 5, 30(44), 10806 - 13
Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes; Ellis J et al.; Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates . Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest . The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method . The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis . The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes . The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity . The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Nov 5, 30(44), 10799 - 805
Intrinsic fluorescence of chloramphenicol acetyltransferase: responses to ligand binding and assignment of the contributions of tryptophan residues by site-directed mutagenesis; Ellis J et al.; Replacement by tyrosine or phenylalanine was used to assign the additive contributions of each of the three tryptophan residues of chloramphenicol acetyltransferase (CAT) to its intrinsic fluorescence on excitation at 295 nm . During the assessment of the fluorescence responses of the wild-type enzyme to the binding of ligands, it was found that the overlapping absorption spectra of chloramphenicol and tryptophan, with an attendant inner filter effect, required the use of a displacement technique involving an alternative substrate (the p-cyano analogue of chloramphenicol) without significant absorption at 295 nm . By the use of two-Trp, one-Trp, and Trp-less variants, in combination with this displacement technique, it was possible to demonstrate that Trp-86 and Trp-152 are involved in the fluorescence quenching associated with the binding of chloramphenicol, most likely via nonradiative energy transfer from these residues to the bound substrate . Trp-152 is mainly responsible for the fluorescence enhancement accompanying the binding of acetyl-CoA (and CoA) through proximity effects and solvent exclusion on substrate association.

Biochemistry, 1991 Nov 5, 30(44), 10722 - 9
Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli; Truong HT et al.; A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli . The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies . Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment . All 24 mutants thus generated are expressed in E . coli . Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity . The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C . Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity . All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded . Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme . All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS . Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Nov 5, 30(44), 10654 - 63
The secondary structure in solution of acyl-coenzyme A binding protein from bovine liver using 1H nuclear magnetic resonance spectroscopy; Andersen KV et al.; Acyl-coenzyme A binding protein from bovine liver and the protein expressed in Escherichia coli by the recombinant gene of this protein have been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy . This protein has, in addition to the ability to bind acyl-coenzyme A, been reported to have several important physiological and biochemical functions . It is known as the diazepam binding inhibitor, as a putative neurotransmitter, as a regulator of insulin release from pancreatic cells, and as a mediator in corticotropin-dependent adrenal steroidogenesis . The only difference between the protein produced by recombinant techniques and the native acyl-coenzyme A binding protein is the N-terminal acetyl group present only in the native protein . The two proteins have 86 amino acid residues and a molecular mass of approximately 10,000 Da . Complete assignment of the 1H nuclear magnetic resonances has been obtained for a major proportion of the amino acid residues (55 residues), and partial assignment has been achieved for the others (31 residues) . Sequential nuclear Overhauser effects have demonstrated that the protein has a secondary structure consisting of four alpha-helices of residues 1-15, 22-35, 52-60, and 68-85 . Furthermore, a large number of long-range nuclear Overhauser effects have been identified, indicating that the assignment given here will provide a basis for a structure determination of this protein in solution by nuclear magnetic resonance spectroscopy.

J Biol Chem, 1991 Nov 5, 266(31), 21090 - 8
Translation of the first gene of the Escherichia coli unc operon . Selection of the start codon and control of initiation efficiency; Schneppe B et al.; The uncI gene is the most poorly expressed gene of the unc operon of Escherichia coli . We examined how the structural features of the translational initiation region (TIR) of this gene relate to the efficiency of its expression . A set of various TIR sequences was created for the uncI gene by coupling synthetic oligodeoxynucleotides to the 5' part of the plasmid-borne gene . Analyses of transcription and translation of the resulting constructs revealed that weak translational initiation efficiency of the uncI TIR sequence is a rate-controlling factor for expression . Moreover, the identification of an endonucleotytic cleavage site within the 5' part of the uncI mRNA by Northern blotting lends further support to the notion that instability of the uncI cistron within the polycistronic unc mRNA represents a further mode of control . The analysis of the roles of the different Shine-Dalgarno regions and putative start codons within the translational initiation region of the uncI gene revealed that the Shine-Dalgarno region III and the GUG codon (codon 4 of the uncI coding sequence described by Walker, J . E., Saraste, M., and Gay, N . J . (1984) Biochim . Biophys . Acta 768, 164-200) define the main site of initiation . However, manipulations of the translational initiation region led to selection of an initiation codon further upstream . The relationship between mRNA sequence and higher order structure on the one hand, and initiation site selection and initiation efficiency on the other, was analyzed.

J Biol Chem, 1991 Nov 5, 266(31), 20934 - 9
Mutation of alanine 24 to serine in subunit c of the Escherichia coli F1F0-ATP synthase reduces reactivity of aspartyl 61 with dicyclohexylcarbodiimide; Fillingame RH et al.; Dicyclohexylcarbodiimide (DCCD) inhibits the activity of the F1F0-H+ ATP synthase of Escherichia coli by reacting with aspartyl 61 in subunit c of the FO sector to form a stable N-acylurea . The segment of chromosomal DNA which codes the subunits of the FO was cloned from four independently isolated DCCD-resistant mutants, and the sequence of the subunit c gene (uncE) was determined . An Ala24 to serine (A24S) substitution was found in the subunit c gene of each mutant . The A24S uncE gene was cloned into the BamHI site of a mutant derivative of plasmid pBR322 . The A24S subunit c conferred DCCD resistance to a variety of recipient E . coli strains when it was overexpressed from this plasmid . A 7-base pair deletion beginning at position 132 of the plasmid vector was responsible for the observed overexpression . Hoppe et al . (Hoppe, J., Schairer, H . U., and Sebald, W . (1980) Eur . J . Biochem . 112, 17-24) had previously shown that mutation of subunit c Ile28 to threonine or valine resulted in DCCD resistance . The DCCD sensitivities of the membrane ATPase of these mutants and the A24S mutant were compared . DCCD sensitivity decreased in the order: wild-type much greater than I27V greater than I28T = A24S . The venturicidin sensitivities of wild-type and mutant membranes were also examined . The membrane ATPase of the I28T and I28V mutants was venturicidin resistant whereas the A24S substitution resulted in a hypersensitivity to inhibition by venturicidin . These results support a model in which subunit c folds in the membrane like a hairpin, where the region of residues 24-28 in transmembrane helix-1 is close to that of aspartyl 61 in transmembrane helix-2.

J Biol Chem, 1991 Nov 5, 266(31), 20828 - 32
Characterization of the functional properties of the 70-kDa protein of Mycobacterium bovis; Peake P et al.; A number of mycobacterial proteins have been shown to induce strong humoral and cellular immune responses, including the 70-kDa antigen (p70) of Mycobacterium leprae and Mycobacterium bovis . On the basis of sequence homology and an ATP binding ability, p70 has previously been tentatively allocated to the 70-kDa family of heat shock proteins (hsp70) . We have purified the M . bovis p70 antigen and described ATPase and Ca(2+)-dependent autophosphorylating activities . These co-purified with p70 on gel chromatography and were up-regulated by native proteins and down-regulated by peptides . Inhibitory peptides were shown to bind p70 . These data imply close functional similarities of mycobacterial p70 to other members of the hsp70 family, the Escherichia coli homologue dnaK in particular.

J Mol Biol, 1991 Nov 5, 222(1), 59 - 66
Specificity of antitermination mechanisms . Suppression of the terminator cluster T1-T2 of Escherichia coli ribosomal RNA operon, rrnB, by phage lambda antiterminators; Ghosh B et al.; Transcription of the ribosomal RNA operons (rrn) in Escherichia coli is subject to an antitermination mechanism whereby RNA polymerase is modified to a termination-resistant form during transit through the rrn leader region . This antitermination mechanism is unable to overcome the T1-T2 terminator cluster located at the end of an rrn operon, such as rrnB . We have tested the specificity with which the T1-T2 terminators override an antitermination mechanism, by placing the terminator cluster downstream from the nut and qut sites recognized by phage lambda N and Q gene antiterminators, respectively . Measurement of downstream gene expression shows that RNA polymerase modified by either N or Q reads through the T1-T2 terminators quite efficiently . This supports the view that T1-T2 are not superterminators, and that the rrn antitermination mechanism may have a restricted terminator specificity.

J Biol Chem, 1991 Nov 5, 266(31), 20803 - 9
Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver; Watanabe S et al.; To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+ . The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively . From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated . In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively . In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively . The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells . The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA . When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased . The results indicate that most polyamines exist as a polyamine-RNA complex in cells . Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.

Biochemistry, 1991 Nov 5, 30(44), 10623 - 31
Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding; Sobol RW et al.; Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated . Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer . In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes . Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT . Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component . Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template . The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing . Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300 . This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction . Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

J Biol Chem, 1991 Nov 5, 266(31), 21215 - 23
Cloning, expression, and protein interaction of human nebulin fragments composed of varying numbers of sequence modules; Jin JP et al.; Nebulin, a family of giant myofibrillar proteins of 600-900 kDa, contains a large number of highly conserved sequence repeats of 31-38 amino acids . To investigate the significance of this repeat, human skeletal muscle nebulin cDNA fragments encoding two, six, seven, eight, or fifteen repeat modules were expressed in high yield as nonfusion proteins in Escherichia coli with the pET3d plasmid vector . F-actin cosedimentation and solid phase binding assays demonstrated that all nebulin fragments, except the smallest two-module 67-mer, bound to muscle actin with high affinity under physiological ionic conditions . Solid phase binding assays also revealed that a six-module fragment, NB5, binds to myosin and C-terminal protein but fails to bind to tropomyosin, troponin, and tubulin . Furthermore, the binding of NB5 to actin was inhibited by both tropomyosin and troponin . Immunoelectron microscopic localization of NB5 indicated that this N-terminal region fragment is situated near the distal end of thin filaments in the sarcomere . These results indicate that nebulin is a giant protein with an unprecedently large number of actin-binding sites along its length and is anchored at the C terminus to the Z line in the sarcomere . Nebulin may function as a multifunctional template protein that regulates the length of thin filaments and participates in muscle activities by interacting with actin and myosin filaments in the sarcomere of skeletal muscles.

J Biol Chem, 1991 Nov 5, 266(31), 20810 - 7
Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli; Stephens CM et al.; The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor . The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper . Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA . The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+ . The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+ . Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine . The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.

Biochemistry, 1991 Nov 5, 30(44), 10738 - 45
Identification and characterization of a new family of high-affinity receptors for Escherichia coli heat-stable enterotoxin in rat intestinal membranes; Hugues M et al.; Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined . The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions . Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein . In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein . Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding . These sites exhibited similar dissociation but markedly different association kinetics . Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively . These data agree closely with those obtained in studies of equilibrium binding . Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin . Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase . In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1991 Nov 5, 266(31), 20976 - 83
A truncated protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed in Escherichia coli; Luo JH et al.; The amino-terminal domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) contains a serine/threonine-specific protein kinase that has characteristics of a growth factor receptor (Chung, T . D., Wymer, J . P., Smith, C . C., Kulka, M., and Aurelian, L . (1989) J . Virol . 63, 3389-3398; Chung, T . D., Wymer, J . P., Kulka, M . Smith, C . C., and Aurelian, L . (1990) Virology 179, 168-178) . To characterize this protein kinase (PK) domain further we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445 . Bacteria containing pJL11 were induced to express a 29-kDa protein (designated pp29la1) that represents a truncated portion of the ICP10-PK domain (includes PK catalytic motifs I-V) as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10-positive eukaryotic cells, and peptide mapping.pp29la1 has autophosphorylating and transphosphorylating activity for calmodulin . The enzyme is activated by Mn2+ but not by Mg2+ ions, and autophosphorylation is inhibited by histone . It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine.

J Biol Chem, 1991 Nov 5, 266(31), 20626 - 35
The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor; Sakano K et al.; Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities . Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e . 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM) . In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively . The affinities of the two other Class I mutants, {Leu27}- and {Leu43}rIGF-IIs, for these two receptors were reduced 80- to 220-fold . The affinities of Class II mutants, i.e . {Thr48,Ser49,Ile50}- and {Arg54,Arg55} rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor . In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations . For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin . In two biological assay systems, i.e . the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.

Biochemistry, 1991 Nov 5, 30(44), 10769 - 77
Selectivity of the beta-adrenergic receptor among Gs, Gi's, and Go: assay using recombinant alpha subunits in reconstituted phospholipid vesicles; Rubenstein RC et al.; The selective regulation of Gs (long and short forms), Gi's (1, 2, and 3), and Go by the beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta gamma subunits, and individual recombinant G-protein alpha subunits that were expressed in and purified from Escherichia coli . Receptor and beta gamma subunits were incorporated into phospholipid vesicles, and the alpha subunits bound to the vesicles stoichiometrically with respect to beta gamma . Efficient regulation of alpha subunit by receptor required the presence of beta gamma . Regulation of G proteins was measured according to the stimulation of the initial rate of GTP gamma S binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange . The assays yielded qualitatively similar results . GDP/GDP exchange was a first-order reaction for each subunit . The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein . Reconstituted alpha s (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs . Both alpha i,1 and alpha i,3 were regulated with 25-33% of that efficiency . Stimulation of alpha o and alpha i,2 was weak, and stimulation of alpha o was barely detectable over its high basal exchange rate . Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.

FEBS Lett, 1991 Nov 4, 292(1-2), 259 - 63
Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase; Fourmy D et al.; Comparison of the amino-acid sequences of several methionyl-tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance . The role of one of these motifs, centered at position 300 in the E . coli enzyme sequence, was assayed by the use of site-directed mutagenesis . Substitution of the His301 or Trp305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val298 into Ala had only a moderate effect . The catalytic rate of the enzyme was unimpaired by these substitutions . It is concluded that the above conserved amino-acid region is located at or close to the amino-acid binding pocket of methionyl-tRNA synthetase.

FEBS Lett, 1991 Nov 4, 292(1-2), 13 - 6
Transcriptional analysis of two Rhodobacter capsulatus ferredoxins by translational fusion to Escherichia coli lacZ; Suetsugu Y et al.; Plasmids which contained the translational fusion of Escherichia coli lacZ to Rhodobacter capsulatus ferredoxin genes, fdxN and fdxA, were constructed . Effects of growth conditions on the expression of each ferredoxin were analyzed by measuring the beta-galactosidase activity in R . capsulatus which harbored a corresponding plasmid . Transcription of fdxN::lacZ, the ferredoxin I fusion gene, was regulated at least 100-fold by either NH4+ or O2 but not by illumination, confirming that fdxN belongs to the nif-gene family . Transcription of fdxA::lacZ, the ferredoxin II fusion gene, however, was constant under all the conditions surveyed, suggesting that the protein has some constitutive function(s).

FEBS Lett, 1991 Nov 4, 292(1-2), 121 - 7
Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain . Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester; Manjarrez-Hernandez HA et al.; Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells . The most prominent was an acidic phosphoprotein(s) of Mr 20-21 kDa . The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein; (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester, TPA, also increased the phosphorylation of the 20 kDa myosin light chain . In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

FEBS Lett, 1991 Nov 4, 292(1-2), 145 - 7
Detection and localization of the i protein in Escherichia coli cells using antibodies; Schneppe B et al.; Using antibodies raised against the purified i protein, the expression of the chromosomal uncI gene was demonstrated . The i protein was identified as a component of the cytoplasmic membrane and shown to be present in preparations of Fo or F1Fo . The protein is not associated with the F1 moiety.

FEBS Lett, 1991 Nov 4, 292(1-2), 254 - 8
Cooperativity in ATP hydrolysis by GroEL is increased by GroES; Gray TE et al.; The kinetics of ATP hydrolysis by the 'molecular chaperone' GroEL and the inhibition of this hydrolysis by GroES have been studied in more detail . It is shown that the hydrolysis of ATP by GroEL is cooperative with respect to ATP with a Hill coefficient of 1.86 (+/- 0.13) . In the presence of GroES, there is an increase in the degree of cooperativity with a Hill coefficient of 3.01 (+/- 0.18) . The observed cooperativity is not due to dissociation of the GroEL oligomer into smaller units but more probably involves structural changes within the GroEL oligomer.

Heart Lung, 1991 Nov, 20(6), 692 - 3
Escherichia coli septic arthritis of a shoulder in a diabetic patient; Barzaga RA et al.; Escherichia coli septic arthritis is rare and usually occurs in patients with underlying systemic disorders . Most commonly the hip joint is involved and the E . coli septic arthritis is caused by an intraabdominal source (e.g., an abscess communicating with the hip joint) . We report the first case of E . coli septic arthritis involving the shoulder joint in a diabetic patient.

Ann Hematol, 1991 Nov, 63(5), 276 - 81
Granulocyte-macrophage colony-stimulating factor in acute non-lymphocytic leukemia before and after chemotherapy; Visani G et al.; The introduction of hematopoietic growth factors into the management of leukemia can influence the outcome of treatment in several ways, depending on the sensitivity and the response of normal and leukemic cells . In this paper we report on the effects of the administration of Escherichia coli-produced, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) in 15 adult patients with acute nonlymphocytic leukemia (ANLL) resistant to first-line treatment or in relapse . GM-CSF was given at a dose of 5-10 micrograms/kg/day as a 6-h i.v . infusion, prior to chemotherapy (CHT) (for 7 days) and after CHT (until evidence of failure or of remission) . In the pre-CHT period there was a clear trend towards an increase of circulating neutrophils (PMN) and/or blast cell count (median 0.3 vs . 1.0 x 10(9)/l for PMN, and 0.5 vs . 2.3 for blast cells) . After chemotherapy, in the patients who achieved complete remission (CR), the median time to a PMN count greater than 0.5 x 10(9)/l and greater than 1 x 10(9)/l was 16 days (range 13-27) and 19 days (range 13-42) respectively . The outcome of treatment was CR for 8/15 (53%), death during induction for 3/15 (20%), and failure for 4/15 (27%) . All failures occurred in patients with an increase of blast cell count during pre-CHT GM-CSF administration . Toxicity and side effects were minor, apart from an acute respiratory syndrome that developed twice in the same patient, at doses of 10 and 3 micrograms/kg/day . These data suggest that investigation of GM-CSF in the treatment of ANLL is worth pursuing, with special attention to GM-CSF effects prior to chemotherapy.

Scand J Immunol, 1991 Nov, 34(5), 619 - 26
Highly efficient expression of proteins encoded by recombinant vaccinia virus in lymphocytes; Alonso JM et al.; Using a recombinant vaccinia virus (VV) that expresses E . coli beta galactosidase (beta-Gal) to infect lymphocytes, we show that enzymometrically or immunologically detectable beta-Gal expression is less pronounced among T cells than among B cells . VV infection caused growth inhibition of B cells, but barely affected T-cell proliferation in vitro . Moreover, the production of infectious viral particles was less pronounced in T lymphocytes . Kinetic studies revealed that after an initial dose-dependent growth inhibition, T cells continued to proliferate without the doubling time being affected by VV infection . Nonetheless, the T cells do express proteins encoded by recombinant VV, such as beta-Gal, or secrete soluble proteins such as interleukin-4, though at a lower efficiency at the per cell level than B lymphocytes . In conclusion, the physiology of T cells appears to be less perturbed by VV than that of B cells, although the virus is capable of directing expression of recombinant genes to T lymphocytes.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9685 - 9
The thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3' thymine-to-cytosine transitions in Escherichia coli; LeClerc JE et al.; We have constructed single-stranded, M13-based vectors that contain a specifically located thymine-thymine pyrimidine-pyrimidone(6-4) UV photoproduct and have used these to estimate the frequency and accuracy of DNA replication past this adduct in uvrA6 cells of Escherichia coli . Both the normal and the Dewar valence photoisomer of the (6-4) adduct were studied . In the absence of SOS induction, vectors carrying the photoproducts were rarely replicated; relative to the lesion-free control, 1.9% of vectors carrying the normal (6-4) isomer produced plaques, and with the Dewar valence isomer the proportion was 0.4% . In SOS-induced cells, these frequencies rose to 22.1% and 12.3%, respectively . The error frequency of replication past the normal isomer in SOS-induced cells was high; in a random sample of 185 progeny phage analyzed, 169 (91%) contained mutations, all of which were targeted . Equally striking, a high proportion of the mutations (158/169; 93%) were of only one type, namely 3' T----C transitions . Both the error frequency and the specificity were much reduced with the Dewar valence isomer; overall, 74/140 (53%) of the phage analyzed were mutant, and of these only 34 (46%) entailed the 3' T----C transition . We speculate that the high error frequency and specificity arise from the formation of a stable T-G base pair, involving hydrogen bonds at O-2 and N-3 in the pyrimidone ring . Potential hydrogen bonds at these sites are coplanar in the normal but not in the Dewar isomer, perhaps explaining the reduced specificity of mutagenesis with the latter adduct.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9668 - 70
Quantitation of prenylcysteines by a selective cleavage reaction; Epstein WW et al.; The allylic thioether bond of the prenylcysteines of prenylated proteins has been shown to be cleaved by 2-naphthol under alkaline conditions to yield substituted naphthopyrans . These products are readily resolved from interfering materials by HPLC and have a strongly absorbing chromophore . Thus, this reaction is suitable for quantitative analysis of prenyl substituents of proteins, and we have examined a number of tissues for their content of prenylcysteines . These amino acids are present in mammalian tissues at a concentration of 0.36-1.4 nmol/mg of protein, with a ratio of geranylgeranylcysteine to farnesylcysteine in the range of 4 to 10 . Prenylcysteines were also found in the cytosolic fraction of two mouse tissues at about one-third the concentration of the whole organ . The level of these modified amino acids was found to be significantly less in a yeast, a fungus, a brown alga, a higher plant, and an insect . Again, geranylgeranylcysteine is predominant . Prenylcysteines were absent from Escherichia coli but present in an archaebacterium . The prenylcysteine content of mammalian tissue is about 1% of that of cholesterol and about equal to that of ubiquinones and dolichols . Calculations indicate that about 0.5% of all proteins are prenylated.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9603 - 7
Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions; Sugiyama JE et al.; The Escherichia coli mannitol permease catalyzes the concomitant transport and phosphorylation of D-mannitol . This 68-kDa protein consists of a membrane-bound, N-terminal domain involved in mannitol binding and translocation and a C-terminal, cytoplasmic domain responsible for mannitol phosphorylation . Secondary-structure prediction methods suggest that the N-terminal half of the permease spans the membrane approximately seven times in alpha-helical segments, but these data cannot conclusively predict the structure . We have used gene fusions between mtlA (encoding the permease) and 'phoA (encoding alkaline phosphatase lacking its signal sequence) to further investigate the topology of the mannitol permease . Initially, fusions were constructed by using a lambda TnphoA vector and in vitro cloning of 'phoA into naturally occurring restriction sites in mtlA . However, the former method gave severe problems with insertion "hot-spots" in our vector systems, and the latter method was limited by the number of useful restriction sites . Therefore, we developed a nested-deletion method for creating mtlA-phoA fusions . 'phoA was first cloned downstream from the part of mtlA encoding the membrane-bound half of the permease . This construct was then treated with the appropriate restriction enzymes and with exonuclease III to create random fusions . An analysis of greater than 40 different fusion clones constructed by these methods provides strong evidence for six membrane-spanning regions in the mannitol permease with three relatively short periplasmic loops and two large cytoplasmic loops in the membrane-bound half of the protein.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9558 - 62
The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding; Yuan HS et al.; The 98-amino acid Fis protein from Escherichia coli functions in a variety of reactions, including promotion of Hin-mediated site-specific DNA inversion when bound to an enhancer sequence . It is unique among site-specific DNA-binding proteins in that it binds to a large number of different DNA sequences, for which a consensus sequence is difficult to establish . X-ray crystal structure analyses have been carried out at 2.3 A resolution for wild-type Fis and for an Arg-89----Cys mutant that does not stimulate DNA inversion . Each monomer of the Fis dimer has four alpha-helices, A-D; the first 19 residues are disordered in the crystal . The end of each C helix is hydrogen bonded to the beginning of helix B' from the opposite subunit in what effectively is one long continuous, although bent, helix . The four helices, C, B', C', and B, together define a platform through the center of the Fis molecule: helices A and A' are believed to be involved with Hin recombinase on one side, and helices D and D' interact with DNA lying on the other side of the platform . Helices C and D of each subunit comprise a helix-turn-helix (HTH) DNA-binding element . The spacing of these two HTH elements in the dimer, 25 A, is too short to allow insertion into adjacent major grooves of a straight B-DNA helix . However, bending the DNA at discrete points, to an overall radius of curvature of 62 A, allows efficient docking of a B-DNA helix with the Fis molecule . The proposed complex explains the experimentally observed patterns of methylation protection and DNase I cleavage hypersensitivity . The x-ray structure accounts for the effects of mutations in the Fis sequence . Those that affect DNA inversion but not DNA binding are located within the N-terminal disordered region and helix A . This inversion activation domain is physically separated in the Fis molecule from the HTH elements and may specify a region of contact with the Hin recombinase . In contrast, mutations that affect HTH helices C and D, or interactions of these with helix B, have the additional effect of decreasing or eliminating binding to DNA.

Mutat Res, 1991 Nov, 264(3), 93 - 6
Location of a mutation in the aspartyl-tRNA synthetase gene of Escherichia coli K12; Sharples GJ et al.; A mutation (tls-1) that confers a temperature-sensitive growth phenotype in Escherichia coli was shown by DNA cloning and sequencing to be an allele of aspS, the gene for aspartyl-tRNA synthetase . The mutation, which lies near minute 41 on the genetic map, was located some 2.3 kb from the 5' end of the ruvAB operon . A DNA fragment encoding the carboxy-terminus of AspRS was found to be sufficient to allow growth of a tls-1 strain at the non-permissive temperature.

Mutat Res, 1991 Nov, 264(3), 117 - 8
Mechanistic studies on DNA photolyase, II . Is the blue enzyme isolated from Escherichia coli a mutant?
Begley TP.
The contrasting properties of the DNA photolyases isolated from Escherichia coli, and from Saccharomyces cerevisiae suggested the possibility that the E . coli enzyme may have suffered mutagenesis as a consequence of the extensive use of ultraviolet irradiation/photoreactivation as a selection technique during the cloning . We have therefore recloned this gene using a UV-independent protocol and confirmed the original sequence.

J Nucl Med, 1991 Nov, 32(11), 2126 - 31
The localization of indium-111-leukocytes, gallium-67-polyclonal IgG and other radioactive agents in acute focal inflammatory lesions; McAfee JG et al.; A variety of radioactive agents, injected directly intravenously have demonstrated foci of inflammation by gamma camera imaging, avoiding the in vitro preparation of labeled leukocytes . This study sought to find out if any of these agents mimicked the biodistribution in abscesses and non-target organs of labeled mixed leukocyte suspensions . Eight different agents were compared with 111In-oxine labeled leukocytes in an acute soft tissue E . coli abscess and an acute arthritic lesion in 24 dogs one day after intravenous administration . These included 67Ga-citrate, human and canine polyclonal immunoglobulin (IgG), rabbit anti-dog polyclonal IgG, serum albumin, monoclonal antibody TNT-1 F(ab')2 against nuclear antigens, 57Co-porphyrin and serum albumin nanocolloid . None of these agents achieved abscess concentrations approaching those obtained with labeled leukocytes, and their abscess/blood and abscess/muscle concentration ratios were considerably lower . No statistically significant differences were found between the different radiolabeled proteins evaluated . The abscess concentration of 99mTc-nanocolloid was much lower than that of other agents, and the results with the oldest agent, 67Ga-citrate, were disappointing in these acute experiments.

J Gen Virol, 1991 Nov, 72 ( Pt 11), 2853 - 6
Detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants; Osman TA et al.; The movement protein of red clover necrotic mosaic virus (RCNMV) was expressed in Escherichia coli as a fusion with a maltose-binding protein using the vector pMAL-cRI and used to produce an antiserum . The RCNMV movement protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the movement protein antiserum . The movement protein could be detected 6 h after inoculation and reached a maximum after 24 h . In contrast, the virus capsid protein, detected in a soluble fraction by immunoblotting using a capsid antiserum, continued to increase for 72 h after inoculation.

J Infect Dis, 1991 Nov, 164(5), 986 - 9
An ELISA for the detection of localized adherent classic enteropathogenic Escherichia coli serogroups; Albert MJ et al.; An ELISA for the detection of classic enteropathogenic Escherichia coli (EPEC) serogroups was developed . It detected EPEC positive for localized adherence (LA) by HeLa cell assay and EPEC positive for EPEC adherence factor (EAF) by DNA probe assay . A specific antiserum was raised with LA+ EPEC strain E2348/69 (serotype O127:H6) by immunizing rabbits and then absorbing the antiserum with its LA- derivative, MAR20 . The absorbed antiserum reacted specifically with all 90 strains of E . coli belonging to eight different EPEC serogroups that were LA+ by HeLa cell assay and EAF+ by DNA probe assay . All E . coli strains including EPEC serogroups that were LA- by HeLa cell assay and EAF- by DNA probe assay were also negative by ELISA . Thus the ELISA is 100% sensitive and specific in detecting LA+ classic EPEC serogroups.

J Bacteriol, 1991 Nov, 173(22), 7395 - 400
Absence of hisT-mediated tRNA pseudouridylation results in a uracil requirement that interferes with Escherichia coli K-12 cell division; Tsui HC et al.; We show that hisT function is required for normal growth of Escherichia coli K-12, since a lack of hisT-mediated pseudouridine tRNA modification causes a uracil requirement that interferes with cell division . We also show that hisT transcription is positively growth rate regulated in exponentially growing bacteria and is induced during the transition from exponential to stationary growth phase.

J Bacteriol, 1991 Nov, 173(22), 7249 - 56
Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria; Sherman MY et al.; Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo . However, the formation of such complexes has not been systematically studied . A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL . When an E . coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound . These HSPs did not bind to a normal protein A column . DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog . The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone . The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK . Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG . The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release . This approach allows a one-step purification of these proteins from E . coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria . Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.

J Bacteriol, 1991 Nov, 173(21), 7042 - 5
Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene; Brede G et al.; The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported . The sequence data indicated that the gene product consists of 284 amino acids . This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.

J Bacteriol, 1991 Nov, 173(21), 7033 - 7
Evidence for interactions between MotA and MotB, torque-generating elements of the flagellar motor of Escherichia coli; Stolz B et al.; Cells that overexpress MotA (encoded on a plasmid derived from pBR322) grow slowly because of proton leakage . We have traced this defect to the coexpression of a fusion protein consisting of 60 amino acids from the N terminus of MotB and 50 amino acids specified by pBR322 . Mutations within the N terminus, known to abolish function when present in full-length MotB, reversed the growth defect . Growth also was normal when MotA was coexpressed with wild-type MotB or with a series of MotB N-terminal fragments.

J Bacteriol, 1991 Nov, 173(21), 6837 - 43
Molecular organization and nucleotide sequence of the recG locus of Escherichia coli K-12; Lloyd RG et al.; The nucleotide sequence of the Escherichia coli K-12 recG gene was determined . recG was identified as an open reading frame located between the spoT operon and the convergent gltS gene . It encodes a polypeptide of 693 amino acids which was identified as a 76-kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after it was labeled with {35S}methionine in maxicells . The sequence determined revealed no obvious promoter . Synthesis of RecG by plasmids carrying the intact gene varied with the orientation of the insert relative to the vector promoter and with the extent of upstream spoT operon sequence included in the construction . It is concluded that recG is the fourth and last gene in the spoT operon, although a possible promoter for independent transcription of spoU and recG was identified near the end of the spoT gene . The primary sequence of RecG revealed that it is related to proteins that act as helicases and has a well-conserved motif identified with ATP binding.

J Bacteriol, 1991 Nov, 173(21), 6790 - 7
Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development; Kimsey HH et al.; The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned . The M . xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil . Electroporation of M . xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus . Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications . These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell . Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid . Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M . xanthus development . The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M . xanthus has pyrimidine salvage pathways . During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium . Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.

J Bacteriol, 1991 Nov, 173(21), 6742 - 8
Cloning and characterization of cutE, a gene involved in copper transport in Escherichia coli; Rogers SD et al.; The copper-sensitive/temperature-sensitive phenotype of the Escherichia coli cutE mutant has been complemented by cloning wild-type genomic DNA into the plasmid vector pACYC184 and selecting transformants on medium containing 4 mM copper sulfate and chloramphenicol . One of these complementing clones, designated pCUT1, contained a 5.6-kb BamHI fragment . This recombinant plasmid transformed cutE, allowing wild-type growth of transformants on medium containing copper sulfate . Complementation of copper sensitivity was assessed by comparing both cell survival at increased copper levels and the results of 64Cu accumulation assays . An EcoRI subclone, 2.3 kb in size, was also shown to complement cutE when cloned in both medium- and high-copy-number vectors and was completely sequenced . This clone was mapped on the E . coli physical map at 705.70 to 707.80 kb . A series of subclones was constructed from pCUT1 and used to show that the large open reading frame of the translated sequence was essential for complementation . This open reading frame has a potential upstream promoter region, ribosome-binding site, and transcriptional terminator and encodes a putative protein of 512 amino acids that contains a region showing some homology to a putative copper-binding site.

J Bacteriol, 1991 Nov, 173(21), 6732 - 41
Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity; Rumbak E et al.; A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates . The gene was expressed in Escherichia coli from its own promoter . The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes . A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases . The B . fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold . The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame . The enzymatic properties of the purified enzyme were investigated . The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.

Infect Immun, 1991 Nov, 59(11), 4001 - 12
Recombinant Escherichia coli clones expressing Chlamydia trachomatis gene products attach to human endometrial epithelial cells; Schmiel DH et al.; To identify Chlamydia trachomatis genes involved in attachment to host cells, a chlamydial genomic library was screened on the basis of binding characteristics by two methods . In the whole-cell screen, individual recombinant Escherichia coli clones were assayed for adherence to eukaryotic cells . In the membrane-binding screen, each recombinant colony of E . coli was treated with CHCl3 and assayed for binding to purified, 3-{(3-cholamidopropyl)-dimethyl-ammonio}-1-propanesulfonate (CHAPS)-solubilized, 35S-labeled eukaryotic membrane material . Initial screening with McCoy cells was refined by using HEC-1B cells, a human endometrial epithelial cell line, which discriminate among recombinants adhering to McCoy cells . Some recombinants demonstrate significantly greater adherence to HEC-1B cells than to McCoy cells and appear, by transmission electron microscopy, to associate with electron-dense areas of the epithelial cell plasma membrane, resembling coated pits . Recombinants positive by one or both screening methods were examined by Southern and Western (immunoblot) analyses, which revealed the presence of chlamydial sequences inserted in the plasmids and the expression of novel 18-, 28-, and approximately 82 kDa, and perhaps of 18 Maxicell analysis of selected recombinants confirmed that the proteins of 28 and approximately 82 kDa, and perhaps of 18 kDa, are plasmid encoded . Antiserum generated against the recombinant approximately 82-kDa protein reacted in Western analysis with a similar-sized protein from C . trachomatis serovar E elementary bodies (EB) and reticulate bodies, serovar L2 EB, and C . psittaci EB . E . coli JM109(pPBW58) contains a 6.7-kb plasmid insert which encodes proteins of all three sizes . Under a number of different conditions in the whole-cell attachment assay--i.e., at 4 degrees C, in Ca(2+)- and Mg(2+)-free medium, in the presence of trypsin or dextran sulfate, and with rabbit aortic endothelial cells--the binding specificity of JM109(pPBW58) parallels that of C . trachomatis EB . Finally, the adherence phenotype of E . coli JM109(pPBW58) correlates directly with the presence of the recombinant plasmid; the phenotype is lost concurrently with loss of the recombinant plasmid, and the into E . coli JM109 . The role of the 18-, 28-, and approximately 82-kDa proteins in mediating attachment, whether they act in concert as a complex or individually, has yet to be determined.

Infect Immun, 1991 Nov, 59(11), 3889 - 94
Single-dose tumor necrosis factor protection against endotoxin-induced shock and tissue injury in rats; Alexander HR et al.; Tumor necrosis factor (TNF), a macrophage product released in response to endotoxin and other stimuli, has been shown to be a central mediator of endotoxin or septic shock . However, its highly conserved and wide-ranging physiological effects suggest that it may also be an essential cytokine in the host defense against acute bacterial infection or sepsis . A single nontoxic dose of human recombinant TNF administered intravenously 24 h prior to a lethal infusion of Escherichia coli lipopolysaccharide (LPS) completely prevented acute LPS-induced hypotension, ameliorated tissue injury in the lungs and liver, and improved survival in male Fisher 344 rats . The protective effects of TNF were dose dependent and required a 24-h pretreatment interval . After the infusion of LPS, animals in both groups (TNF-treated animals and saline-pretreated controls) initially appeared acutely ill and had a similar severe metabolic acidosis, indicating that TNF did not inactivate or prevent the toxic effects of LPS . Twelve hours after the administration of TNF, the gene for manganous superoxide dismutase, a mitochondrial enzyme which scavenges toxic reactive oxygen species and is induced during conditions which generate a free radical stress, was expressed in liver tissue, suggesting that the induction of manganous superoxide dismutase may be an important in vivo protective mechanism against cellular injury during lethal endotoxemia.

Infect Immun, 1991 Nov, 59(11), 3869 - 75
Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli; Jerse AE et al.; Attaching and effacing (A/E) intestinal lesions are produced by enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E . coli (EHEC), and RDEC-1, a pathogen of weanling rabbits . We recently identified a chromosomal locus (eae{E . coli A/E}) which is required for A/E activity in a wild-type EPEC strain . Sequences homologous to those of an eae gene probe were detected in EPEC, RDEC-1, and EHEC isolates . We report here that the eae gene is chromosomally encoded in all EPEC and EHEC strains tested and in RDEC-1 . In addition, the eae probe was found to be 100% sensitive and 98% specific in detecting E . coli of EPEC serogroups that demonstrate A/E activity . Ten percent of E . coli of EPEC serogroups that hybridized with the eae probe and produced A/E activity did not hybridize with the EAF (EPEC adherence factor) probe, a plasmid-associated diagnostic probe which is currently used to identify EPEC . In addition to A/E factors, plasmid-associated adhesins also contribute to the pathogenesis of EPEC and RDEC-1 . To further investigate the role of plasmid-associated adherence, a hybrid RDEC-1-EPEC strain containing the adherence plasmid of an EPEC strain in the A/E background of RDEC-1 was constructed . This hybrid strain, unlike the parent RDEC-1 strain, produced A/E lesions on human tissue culture cells, which suggests that the EPEC adherence plasmid provides tissue specificity to the hybrid strain and that the A/E factors of RDEC-1 are not host restricted.

Genes Dev, 1991 Nov, 5(11), 1946 - 56
The mammalian TFIID protein is present in two functionally distinct complexes; Timmers HT et al.; The TFIID activity recognizes a TATA-box element and supports formation of an initiation complex containing RNA polymerase II . Antisera specific for the 38-kD human TFIID protein were used to determine whether this protein cofractionated with the TFIID activity . Surprisingly, the TFIID activity in HeLa whole-cell extracts was resolved into two different size complexes, one of 300 kD and one of greater than 700 kD . Cofractionation studies suggest that both complexes contain the 38-kD protein; thus, this component of the large complexes is probably responsible for recognition of the TATA sequence and interaction with the other general transcription factors in formation of the initiation complex . Interestingly, in contrast to the TFIID activity characterized previously, the 300-kD form of TFIID activity, B-TFIID, does not support stimulation of transcription by factors containing acidic or glutamine-rich activating motifs . We propose that the functional and physical differences between these two forms of TFIID activity are caused by differences in the protein composition of the TFIID complexes of which the 38-kD hTFIID protein is an integral part.

Dev Biol, 1991 Nov, 148(1), 107 - 16
The cloning and characterization of a maternally expressed novel zinc finger nuclear phosphoprotein (xnf7) in Xenopus laevis; Reddy BA et al.; We report the cloning of a cDNA (xnf7) coding for a maternally expressed Xenopus protein that becomes highly enriched in nuclei of the central nervous system during later development and in nuclei of adult brain . The protein also shows stage-specific nuclear/cytoplasmic partitioning and phosphorylation that may be related to its function . In addition, it binds to double-stranded DNA in vitro . The conceptual protein produced by the xnf7 clone contains several acidic domains, a novel zinc finger domain, three putative p34cdc2 protein kinase phosphorylation sites, and a bipartite basic nuclear localization signal . The xnf7 mRNA was detected as a maternal transcript that decreased in abundance during development through the gastrula stage . It was reexpressed at the neural stage in mesoderm and neural tissues, and its reexpression was not dependent upon the normal juxtaposition of the mesoderm and ectoderm that occurs during neural induction as demonstrated by high titer in exogastrulae . In situ hybridization showed enrichment of the mRNA in the neural tube and a small amount in the mesoderm at the late neurula stage . Xnf7 is normally phosphorylated during oocyte maturation . The bacterially expressed xnf7 protein was phosphorylated in vitro by purified maturation-promoting factor at a threonine in a small N-terminal domain containing one of the p34cdc2 protein kinase phosphorylation sites, but not by several other protein kinases . The structural domains present in the protein and its localization in nuclei suggest that the xnf7 gene product performs an important nuclear function during early development, perhaps as a transcription factor or a structural component of chromatin.

Eur J Biochem, 1991 Nov 1, 201(3), 569 - 79
NMR studies of the activation of the Escherichia coli trp repressor; Hyde EI et al.; The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis . Tryptophan analogues lacking the alpha-amino group act as inducers of trp operon expression . We have used one- and two-dimensional 1H-NMR spectroscopy to compare the binding to the repressor of the corepressors L-tryptophan, D-tryptophan and 5-methyl-DL-tryptophan with that of the inducer indole-3-propionic acid . We have determined the chemical shifts of the indole ring protons of the ligands when bound to the protein, principally by magnetization-transfer experiments . The chemical shifts of the indole NH and C4 protons differ between corepressors and inducer . At the same time, the pattern of intermolecular NOE between protons of the protein and those of the ligand also differ between the two classes of ligand . These two lines of evidence indicate that corepressors and inducers bind differently in the binding site, and the evidence suggests that the orientation of the indole ring in the binding site differs by approximately 180 degrees between the two kinds of ligand . This is in contrast to a previous solution study {Lane, A.N . (1986) Eur . J . Biochem . 157, 405-413}, but consistent with recent X-ray crystallographic work {Lawson, C.L . & Sigler, P.B . (1988) Nature 333, 869-871} . D-Tryptophan and 5-methyltryptophan, which are more effective corepressors than L-tryptophan, bind similarly to L-tryptophan . The indole ring of D-tryptophan appears to bind in essentially the same orientation as that of the L isomer . There are, however, some differences in chemical shifts and NOE for 5-methyltryptophan, which indicate that there are significant differences between the two corepressors L-tryptophan and 5-methyltryptophan in the orientation of the indole ring within the binding site.

Eur J Biochem, 1991 Nov 1, 201(3), 561 - 8
The catalytic domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli . Expression, purification, properties and preliminary X-ray analysis; Schulze E et al.; Partial sequences of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E . coli TG2 . A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine-proline-rich sequence between a putative binding region for the peripheral components and the catalytic domain (apa-4) . Most of the catalytic domain from A . vinelandii without the apa-4 sequence was degraded intracellularly, probably due to incorrect folding . Fusion proteins of six amino acids from beta-galactosidase, the apa-4 region and the catalytic domains of A . vinelandii or E . coli E2p could be highly purified . Both catalytic domains were assembled in 24-subunit structures with a molecular mass of approximately 670 kDa . The expression of catalytic domain from A . vinelandii E2p is more than twice as high as found for wild-type E2p . This can be explained by intracellular degradation of over-expressed wild-type E2p, whereas the catalytic domains are stable against proteolysis in vivo and in vitro . The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose-6 and sedimentation velocity experiments . No binding of either E1p or E3 to the catalytic domain of either organism was detectable . Crystals of the catalytic domain of A . vinelandii E2p could be grown to a maximum size of 0.6 x 0.6 x 0.4 mm . They diffract up to a resolution of 0.28 nm.

Dig Dis Sci, 1991 Nov, 36(11), 1562 - 8
Misoprostol but not antacid prevents endotoxin-induced gastric mucosal injury: role of tumor necrosis factor-alpha; Mahatma M et al.; Many of the complications of septic shock are believed to be a consequence of elevated circulating levels of tumor necrosis factor (TNF), which is an important mediator of tissue injury . Prostaglandins (PGs) of the E series have recently been reported to inhibit TNF production in vitro . We investigated the in vivo effect of misoprostol, a PGE1 analog, on endotoxin-induced gastric mucosal injury and TNF production . For the gastric mucosal injury studies, groups of animals were pretreated with intragastric misoprostol (100 and 200 micrograms/kg) or with antacid (2 ml/animal of Maalox Plus) 30 min prior to a challenge with intravenous E . coli lipopolysaccharide (LPS) at 5.0 mg/kg . Stomachs were examined 3 hr after LPS . Systemic endotoxin alone induced microscopic edema, vascular congestion, and polymorphonuclear (PMN) infiltration of the gastric mucosa . Pretreatment with misoprostol, but not with antacid, significantly and dose-dependently reduced the gastric mucosal injury . For the TNF studies, groups of rats were given either misoprostol (100 or 200 micrograms/kg, intragastric), or saline 1 hr prior to LPS challenge . Serum samples were obtained 1.5 hr after LPS challenge . Misoprostol dose-dependently and significantly (P less than 0.01) inhibited TNF activity . We conclude that misoprostol is a potent inhibitor of TNF systemic production and inhibits the gastric mucosal injury induced by endotoxemia . These studies suggest a potentially important therapeutic role for misoprostol in inflammatory diseases in which TNF exerts a contributory role.

Cancer Res, 1991 Nov 1, 51(21), 5843 - 50
Nitrosamine-induced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras; Georgiadis P et al.; Mammary and skin tumors induced in rodents by N-methyl-N-nitrosourea treatment have a G:C to A:T transition mutation in codon 12 of H-ras, probably resulting from alkylation of O6 of guanine by the carcinogen . This codon contains two guanines (5'-GGA-3'), but mutations are observed only in the central base pair of this codon . The same selectivity for mutations of -GGA-sequences has also been observed in Escherichia coli . It is known that the central G in the sequence GGA is a preferred site for alkylation, but the magnitude of chemical selectivity is insufficient to provide a complete explanation for the biological observation which is still unexplained . We have measured accurate rates of repair by the E . coli and gene O6-alkylguanine-DNA-alkyltransferase of an O6-methylguanine in various positions in chemically synthesized 15-base pair DNA duplexes having the H-ras sequence . The rate of repair varied 25-fold, depending on the sequence flanking the methylguanine . An O6-methylguanine in position 2 of codon 12 was the least well repaired . The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation for the selective mutation of this position . Using an antibody to probe the accessibility of the O6-methyldeoxyguanosine, it was shown that the rate of repair is a reflection of the conformation of the sequence containing the alkylated base, because the avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the methylguanine, with the most rapidly repaired O6-methylguanines being those most easily bound by the antibody.

Plant Mol Biol, 1991 Nov, 17(5), 973 - 83
Sugar response element enhances wound response of potato proteinase inhibitor II promoter in transgenic tobacco; Kim SR et al.; The promoter region of the potato proteinase inhibitor II (PI-II) gene was studied to identify cis-acting regulatory sequences involved in sugar response using transgenic tobacco plants . The 5' control region covering an 892 nucleotide sequence upstream from the cap site and a 32 nucleotide untranslated region of the PI-II promoter was able to activate a reporter chloramphenicol acetyltransferase (cat) gene by wounding or by incubating in a sugar-free medium . This wound response was further enhanced by sugar . Hexoses, disaccharides, and some trisaccharides were strong inducers whereas pentoses, deoxy sugars, sugar acids, TCA cycle intermediates, amino acids, and other carbohydrates had little effect on the promoter activity . Deletion of the sequence between -892 and -573 abolished the wound response but not the sugar response . An additional 5' deletion to -453 removed the sugar inducibility . Locations of the cis-acting regulatory elements were further elucidated by 3' deletion analysis . Deletion of the downstream region from -520 did not affect the wound or sugar response of the promoter . However, 3' deletion mutant -574 was unable to respond to sugar but did respond weakly to wounding . Further deletion to -624 abolished both responses . Therefore, it can be concluded that a wound response element is located in between -624 and -574 and that the response is further enhanced by a sugar response element located in the sequence between -573 and -520.

Plant Mol Biol, 1991 Nov, 17(5), 1005 - 11
Site-directed mutagenesis and expression in Escherichia coli of WMAI-1, a wheat monomeric inhibitor of insect alpha-amylase; Garcia-Maroto F et al.; The wheat monomeric inhibitor WMAI-1 (syn . 0.28) produced in Escherichia coli using the pT7-7 expression vector has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the alpha-amylase from the insect Tenebrio molitor as the native WMAI-1 isolated from wheat . This confirms that the native inhibitor is not glycosylated and contradicts claims that a putative glycosyl moiety was essential for inhibition . Thirteen mutants have been obtained at six different sites . Substitution of the highly conserved N-terminal S by the sequence ARIRAR increased the pre-incubation time required for maximum activity . A similar result was obtained by insertion of GPRLPW after position 4, while insertion of EPRAPW at the same position rendered the inhibitor inactive . The substitution D/EGPRL and insertions DGP or D, at position 58, produced complete inactivation . All other mutations had only minor effects on activity.

Arch Biochem Biophys, 1991 Nov 1, 290(2), 376 - 80
Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli; Wada A et al.; Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E . coli strain JM109 and transcription induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) . Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases . The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E . coli (40-60 nmol/liter culture) . Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E . coli . The P450scc was found to be associated with the E . coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450) . This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.

Arch Biochem Biophys, 1991 Nov 1, 290(2), 336 - 44
Characterization of a cDNA-encoding rabbit brain heme oxygenase-2 and identification of a conserved domain among mammalian heme oxygenase isozymes: possible heme-binding site?
Rotenberg MO, Maines MD.
A 1.3-kb rat testis cDNA clone for heme oxygenase-2 (HO-2) was used as a Northern blot hybridization probe, and a single homologous mRNA species, of approximately 1.3 kb in rabbit brain and testis was detected . This contrasted with the observation made with rat brain in which two HO-2 transcripts of approximately 1.3 and 1.9 kb were detected . Use of the same rat HO-2 probe to screen a rabbit brain cDNA library in lambda gt11 resulted in the recovery of a single 1.2-kb cDNA clone . This cDNA exhibits 84% overall nucleotide sequence homology with rat HO-2 and encodes a protein of 35,352 Da, displaying 88% amino acid sequence homology with rat testis HO-2 . Furthermore, when expressed in Escherichia coli, the rabbit cDNA-encoded protein displays heme oxygenase activity and cross-reactivity with antibody to rat HO-2 . Based on findings obtained through Western immunoblot analysis of partially purified HO-2 protein prepared from rabbit testis and brain, the 35- to 36-kDa molecular form appears to be the major HO-2 form detected in the brain, whereas a 42-kDa species is the predominant form observed in rabbit testis . Having deduced the amino acid sequence of rabbit brain HO-2, we provide a comparison of this sequence with those of rat, mouse, and human HO-1 and rat HO-2, and thereby identify a 24-amino-acid-long peptide region which, except for one residue, is identical in all five species of HO-1 and HO-2 compared (96% similarity), and exhibits 100% similarity in predicted secondary structure (for this region) in all five proteins . We propose that this peptide may be important to the heme binding and isomer-specific tetrapyrrole cleavage activities of the heme oxygenase isozymes.

Virology, 1991 Nov, 185(1), 493 - 5
cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 are rendered biologically active in a plasmid containing the cauliflower mosaic virus 35S promoter; Commandeur U et al.; cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 could be rendered biologically active when they were placed under the control of the cauliflower mosaic virus 35S promoter and polyadenylation signal . Although the 35S in vivo transcripts should have contained up to forty 5' and several hundred 3' nonviral nucleotides, the progeny viral RNAs had the same sizes as in naturally infected sugarbeets . The progeny RNAs did not hybridize with the nonviral sequences indicating that they were apparently not replicated . Deletion and insertion mutants of RNA 3 cDNA clones were also biologically active in plants but a plasmid which contained the cDNA of RNA 3 in antisense orientation was not . The biological activity of plasmid DNAs compared with the corresponding synthetic transcripts is discussed.

Virology, 1991 Nov, 185(1), 354 - 64
Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses; Qu RD et al.; Rice tungro disease is caused by an infection of two different viruses, rice tungro spherical virus (a (+) sense RNA virus) and rice tungro bacilliform virus (RTBV) with a genome of circular double-stranded DNA . The genome of an RTBV isolate from the Philippines was cloned, sequenced, and found to be 8000 bp in length . It contains four open reading frames (ORFs) on a single strand, with ORF 1 having an internal termination codon (TAA) . The 5' and 3' ends of a polyadenylated viral RNA transcript, of genome length, were mapped by primer extension and cDNA sequence analysis, respectively . The transcript is terminally redundant by 265-268 nucleotides . Purified virus particles contain two major proteins with molecular masses of 37 and 33 kDa, although only the 37-kDa protein was detected in the infected rice tissues . The N-terminal amino acid sequence of the 33-kDa protein was determined and its coding region was identified on the RTBV genome . The identity of the coat protein gene was further confirmed by expressing a region of the genome in Escherichia coli, the products of which reacted with anti-RTBV antibody . The unusually long ORF 3 of RTBV is predicted to encode a polyprotein of 194.1 kDa that includes: the coat protein(s), viral proteinase, reverse transcriptase, and ribonuclease H . The sections of the polyprotein show varying degrees of similarity to the counterparts of Commelina yellow mottle virus (a member of the proposed badnavirus group) and caulimoviruses . The functions of the other three ORFs are unknown.

Virology, 1991 Nov, 185(1), 32 - 8
Structure of the glycoprotein gene of sonchus yellow net virus, a plant rhabdovirus; Goldberg KB et al.; The nucleotide sequence of the glycoprotein (G) gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined from viral genomic and mRNA cDNA clones . The G cistron is 2045 nucleotides (nt) long and the G protein mRNA open reading frame (ORF), as determined from the cDNA sequence, contains 1896 nt and encodes a protein of 632 amino acids . Immunoblots with antibodies elecited against the purified glycoprotein from virus particles reacted with a fusion protein produced in Escherichia coli, indicating that the cloned ORF encodes the G protein . The 5' end of the G protein mRNA corresponds to nt 5111, relative to the 3' end of the viral (minus sense) genome, as determined by primer extension from mRNA isolated from infected plants, and extends to nt position 7155 on the genomic RNA . A 34-nt untranslated 5' leader sequence and a 115-nt untranslated 3' end flank the ORF on the mRNA . The gene junctions on either side of the G gene on the genomic RNA are identical to those previously described for other SYNV genes and are similar to sequences separating genes of animal rhabdoviruses . The predicted molecular weight of the G protein is 70,215 Da, a value less than the 77,000 Da estimated for the glycosylated G protein from virus particles . The deduced amino acid sequence of the SYNV G protein shares little direct relatedness with the G proteins of other rhabdoviruses, but appears to contain a similar signal sequence, a transmembrane anchor domain, and glycosylation signals . In addition, the SYNV G protein contains a putative nuclear targeting site near the carboxy terminus, which may be involved in transit to the nuclear membrane prior to morphogenesis.

Virology, 1991 Nov, 185(1), 242 - 50
Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae; Palm P et al.; The DNA sequence of the Sulfolobus shibatae virus SSV1 is the first complete sequence of an archaebacterial virus genome . The viral DNA is a closed double-stranded DNA circle of 15465 bp . The features of the sequence, the positions of all 11 transcripts, the three characterized proteins, and the open reading frames are described.

Virology, 1991 Nov, 185(1), 18 - 31
Effects of random mutagenesis upon potato spindle tuber viroid replication and symptom expression; Owens RA et al.; A combination of random chemical mutagenesis plus temperature gradient gel electrophoresis was used to isolate a collection of 57 potato spindle tuber viroid (PSTV) cDNAs containing mutations distributed throughout the entire 359 nucleotide genome . Although the presence of multiple mutations was often associated with a loss of cDNA infectivity, infectious PSTV cDNAs containing as many as four unlinked alterations could be isolated . Several mutations in the pathogenicity domain and left terminal loop were stably maintained in the resulting progeny, but those which affect base pairing in secondary hairpins I and II were either lethal or rapidly reverted to wild-type . One stable C----U substitution which may promote significant structural rearrangement within the right side of the pathogenicity domain had no detectable effect upon symptom expression . The variable domains of several noninfectious mutants contained an A----G substitution which is likely to inhibit the in vitro formation of secondary hairpin II via stabilization of the native structure, and the lethal nature of this mutation was confirmed by oligonucleotide-directed mutagenesis . Several lines of evidence now point toward an essential role for secondary hairpin II in the replication of PSTV and related viroids.

J Virol, 1991 Nov, 65(11), 6101 - 10
Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor; Zhang YF et al.; A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y . Zhang and B . Moss, Proc . Natl . Acad . Sci . USA 88:1511-1515, 1991) . Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times . Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S . Y . Kao and W . R . Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene . In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal . Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with {35S}methionine or {32P}phosphate . Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation . Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion . Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.

J Virol, 1991 Nov, 65(11), 5721 - 31
The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes: characterization of reovirus core protein sigma 2; Dermody TS et al.; The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes were determined to gain insight into the structure and function of the S2 translation product, virion core protein sigma 2 . The S2 sequences of the type 1 Lang, type 2 Jones, and type 3 Dearing strains are 1,331 nucleotides in length and contain a single large open reading frame that could encode a protein of 418 amino acids, corresponding to sigma 2 . The deduced sigma 2 amino acid sequences of these strains are very conserved, being identical at 94% of the sequence positions . Predictions of sigma 2 secondary structure and hydrophobicity suggest that the protein has a two-domain structure . A larger domain is suggested to be formed from the amino-terminal three-fourths of sigma 2 sequence, which is separated from a smaller carboxy-terminal domain by a turn-rich hinge region . The carboxy-terminal domain includes sequences that are more hydrophilic than those in the rest of the protein and contains sequences which are predicted to form an alpha-helix . A region of striking similarity was found between amino acids 354 and 374 of sigma 2 and amino acids 1008 and 1031 of the beta subunit of the Escherichia coli DNA-dependent RNA polymerase . We suggest that the regions with similar sequence in sigma 2 and the beta subunit form amphipathic alpha-helices which may play a related role in the function of each protein . We have also performed experiments to further characterize the double-stranded RNA-binding activity of sigma 2 and found that the capacity to bind double-stranded RNA is a property of the sigma 2 protein of prototype strains and of the S2 mutant tsC447.

J Cell Biol, 1991 Nov, 115(3), 597 - 605
Three-dimensional reconstruction of the 70S Escherichia coli ribosome in ice: the distribution of ribosomal RNA; Frank J et al.; A reconstruction, at 40 A, of the Escherichia coli ribosome imaged by cryo-electron microscopy, obtained from 303 projections by a single-particle method of reconstruction, shows the two subunits with unprecedented clarity . In the interior of the subunits, a complex distribution of higher mass density is recognized, which is attributed to ribosomal RNA . The masses corresponding to the 16S and 23S components are linked in the region of the platform of the small subunit . Thus the topography of the rRNA regions responsible for protein synthesis can be described.

Exp Cell Res, 1991 Nov, 197(1), 8 - 11
Uptake of hyaluronan in hepatic endothelial cells is not directly affected by endotoxin and associated cytokines; Fraser JR et al.; The uptake of hyaluronan (HYA) labeled with 3H in its acetyl group was measured in cultured liver endothelial cells from normal rats and from rats previously treated with sublethal doses of Escherichia coli endotoxin (ET) . Replicate cultures were also exposed to recombinant human tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or interferon-gamma for 1 to 3 h before the measurement of hyaluronan uptake . Under all conditions, HYA was absorbed by endothelial cells at rates consistent with receptor-mediated absorption . In cells exposed to HYA 20 h after isolation, rate of uptake was less than half the rate in cells exposed 6 or 7 h after isolation . Cellular uptake of HYA was neither reduced nor enhanced by any of the treatments with cytokines . Prior exposure of the cell donors to ET caused a three-fold increase in their plasma HYA but did not alter the subsequent rate of cellular HYA uptake in vitro, either with or without added treatment with TNF-alpha or IL-1 . It was concluded that the elevation of plasma HYA caused by septicaemia or by the experimental administration of ET or TNF-alpha cannot be attributed to direct interference with HYA receptors on hepatic endothelial cells.

EMBO J, 1991 Nov, 10(11), 3495 - 501
Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility; Durrenberger F et al.; All chloroplast 23S ribosomal RNA genes of the unicellular alga Chlamydomonas reinhardtii contain an 888 bp group I intron with an internal open reading frame (ORF) . A precursor RNA encompassing the intron with its 5' and 3' flanking sequences was shown to self-splice both during in vitro transcription and upon incubation of the isolated pre-RNA under self-splicing conditions . Expression of the internal ORF in Escherichia coli in the presence of a plasmid containing a cDNA corresponding to the intronless form of the 23S rRNA gene resulted in specific cleavage of the cDNA at or close to the exon junction sequence . To test whether this ORF-encoded double-strand DNA endonuclease is involved in intron mobility in vivo, the same ribosomal cDNA was stably integrated into the C . reinhardtii chloroplast genome using particle gun mediated transformation . All the transformants with the cDNA integrated at the expected site in the chloroplast genome had the intron precisely inserted at the artificial exon junction site . These experiments demonstrate that the chloroplast ribosomal intron of C . reinhardtii behaves as a ribozyme in vitro and also as a mobile genetic element in vivo provided a target site is present.

EMBO J, 1991 Nov, 10(11), 3363 - 72
A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli; Wang XD et al.; Cell division in Escherichia coli requires the products of the ftsQ, ftsA and ftsZ genes . It is not known how the cell regulates the cellular concentrations of these essential elements of the division system . We describe here a factor that activates cell division by specifically increasing transcription from one of the two promoters that lie immediately upstream of the ftsQAZ gene cluster . The trans-acting factor is the product of the sdiA gene, which was isolated on the basis of its ability to suppress the division inhibitory effect of the MinC/MinD division inhibitor . In addition, the sdiA gene product suppressed the action of other chromosomally encoded division inhibitors, induced minicell formation in wild type cells, and restored division activity to an ftsZ temperature-sensitive mutant grown under nonpermissive conditions . All of these properties were explained by the ability of the sdiA gene product specifically to increase transcription of the ftsQAZ gene cluster, resulting in an increase in cellular concentration of the FtsZ protein . The sdiA gene product is the first factor thus far identified that specifically regulates expression of this key group of cell division genes.

EMBO J, 1991 Nov, 10(11), 3273 - 80
Sequential action of mitochondrial chaperones in protein import into the matrix; Manning-Krieg UC et al.; Translocation and folding of proteins imported into mitochondria are mediated by two matrix-localized chaperones, mhsp70 and hsp60 . In order to investigate whether these chaperones act sequentially or in parallel, we studied their interaction with newly imported precursor proteins in isolated yeast mitochondria by coimmunoprecipitation . All precursors bound transiently to mhsp70 . Release from mhsp70 required hydrolysis of ATP and did not immediately generate a tightly folded protein . For example, after imported mouse dihydrofolate reductase (a soluble monomeric enzyme) had been released from mhsp70, folding to a protease resistant conformation occurred only after a lag and was much slower than the release . Under standard import conditions, no significant association of DHFR with hsp60 could be detected . Similarly, newly imported hsp60 subunit was released from mhsp70 as an incompletely folded, unassembled intermediate which accumulated at low temperature and assembled to hsp60 14-mer at higher temperature in an ATP-dependent manner . Mas2p (the larger subunit of the MAS-encoded processing protease) first bound to mhsp70, then to hsp60, and only then assembled with its partner subunit, Mas1p . We propose that ATP-dependent release from mhsp70 is insufficient to cause folding of imported proteins and that assembly of hsp60 and Mas2p requires sequential, ATP-dependent interactions with mhsp70 and hsp60.

Plant J, 1991 Nov, 1(3), 327 - 32
Characterization of the auxin-regulated par gene from tobacco mesophyll protoplasts; Takahashi Y et al.; The auxin-regulated par gene from tobacco mesophyll protoplasts was characterized in detail to deduce its possible function . An homology search of the par gene in the NBRF databases revealed that the par gene has homology to the stringent starvation protein (ssp) gene of Escherichia coli, which is induced under starved conditions and binds in an equimolar ratio to a holoenzyme of RNA polymerase . Hence, it is supposed that the par gene product could play a similar role to that of ssp . Although sequence homology of the par gene to the Gmhsp 26-A gene from soybean was observed, both genes were shown to respond differently to plant hormones and stresses . Gmhsp 26-A is induced by heat shock, 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinin and abscisic acid (ABA), whereas the par gene was induced only by auxins . Furthermore, cycloheximide treatment prevents 2,4-D-mediated accumulation of Gmhsp 26-A mRNA, but not that of par mRNA . Both par and Gmhsp 26-A respond to CdCl2, but splicing of the par pre-mRNA proceeded in a normal way, whereas splicing off the Gmhsp 26-A pre-mRNA was inhibited . Hence, the par and Gmhsp 26-A genes should have a common ancestor, but have evolved in different directions . Detailed time-course experiments confirmed that the par gene was induced immediately after the addition of auxin and expressed upon the initiation of meristematic activity in tobacco mesophyll protoplasts . As the par gene was induced by the sole treatment of cycloheximide, it was proposed that the par gene belongs to a category of 'superinduction' genes.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biomol NMR, 1991 Nov, 1(4), 349 - 61
Segmental differences in the stability of the trp-repressor peptide backbone; Czaplicki J et al.; Exchange lifetimes of amide protons in trp-repressor with and without the corepressor, L-tryptophan, were studied by heteronuclear 2D NMR spectroscopy . The amide proton exchange times revealed pronounced differences in the stability of different regions of the trp-repressor . The dimeric core of the molecule is relatively compact and homogeneous in terms of the measured parameters in both apo- and holorepressors . On the other hand the DNA-binding region appears less stable and more susceptible to the exchange of its backbone protons with the solvent . The NMR findings reported here are consistent with and amplify information on the stability of the trp-repressor obtained by other methods.

J Cell Biol, 1991 Nov, 115(3), 665 - 75
Primary structure and domain organization of human alpha and beta adducin; Joshi R et al.; Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues . The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs . The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication . Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids . The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM . The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins . The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein . Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail . Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb . In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression . A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin . The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.

Infect Immun, 1991 Nov, 59(11), 3952 - 61
Coccidioides immitis fractions which are antigenic for immune T lymphocytes; Kirkland TN et al.; The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity . We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response . The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays . The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components . The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay . Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive . In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C . immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs) . The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components . The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined . The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C . immitis . Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption . The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF . The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C . immitis . Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way . The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C . immitis.

Biopolymers, 1991 Nov, 31(13), 1565 - 79
A probabilistic model for genetic recombination of nonreplicating lambda-phage DNA, stimulated by "mismatch repair" of UV photoproducts; Hays JB et al.; Genetic recombination of nonreplicating phage lambda-DNA, during infection of homoimmune lysogenic bacteria, was previously observed to be dramatically stimulated by prior uv irradiation of the phages, even when the Escherichia coli hosts lacked the major uv-photo-product excision-repair system (UvrABC) . UvrABC-independent recombination of circular phage molecules depends on host MutHLS functions and on undermethylation of adenines at GATC sites in the phage DNA, and thus appears to be the result of "mismatch repair" of uv photoproducts . Recombinant frequencies pass through a relatively sharp maximum at 20 J/m2 and decrease at higher doses, whereas most plausible models for the process predict monotonic increases with dose, or a plateau at high uv doses . A uv-dose-dependent loss of biological activity (restriction) of all intracellular phage DNA was also observed previously . In order to provide a framework for testing possible explanations for the unusual recombinant-frequency vs uv-dose curve, a statistical model was constructed . This model includes probability terms for all possible one-exchange and two-exchange recombination processes, and incorporates the assumption that dimer recombinants are more susceptible to restriction than monomer parents (or recombinants), because of their larger target size . By adjustment of model parameters, particularly epsilon, the efficiency per photoproduct of initiation of a recombinational exchange, a theoretical dose-response curve that agreed well with experiment was obtained . The best fit corresponded to epsilon = 0.035, close to the previously observed restriction efficiency of 0.053 . In the calculations, the value for h0, the average length of heteroduplex DNA, was taken to be 0.5 lambda units, i.e., about 25 kilobase pairs . This estimate for h0 was obtained here by analysis of the density distributions of the progeny of crosses between nonreplicating density-labeled lambda-phage chromosomes, published by others {M . S . Fox, C . S . Dudney and E . J . Sodergren (1979) Cold Spring Harbor Symposium on Quantitative Biology, Vo . 43, pp . 999-1007}.

Vet Microbiol, 1991 Nov, 29(3-4), 309 - 18
Immunization of pigs with a purified Shiga-like toxin II variant toxoid; MacLeod DL et al.; Passive transfer of neutralizing antibodies and active immunization with a toxoid of purified Shiga-like toxin II variant (SLT-IIv, edema disease toxin) were used to protect pigs against challenge with SLT-IIv . Six 6-week-old pigs were passively immunized by intraperitoneal administration of an immunoglobulin preparation from porcine antiserum against purified SLT-IIv . Six 6-week-old pigs and twelve 2-week-old pigs were actively immunized by two intramuscular injections of 25 micrograms of SLT-IIv toxoid given 2 weeks apart . The 24 immunized pigs and an equal number of age-matched unimmunized control pigs were all challenged by intravenous injection of purified SLT-IIv (6 ng/kg body weight) . The six passively immunized pigs acquired neutralizing SLT-IIv antibody titers of 1280 or 2560 and the 18 actively immunized pigs mounted a serum immune response with toxin neutralizing titers ranging from 128 to 10,240 . All 24 immunized pigs survived the challenge but nine pigs with the lowest titers showed mild clinical signs of edema disease . All 24 control pigs died within 30 hours of challenge.

Virus Res, 1991 Nov, 21(3), 237 - 47
Construction and uses of cell lines containing integrated adenovirus E2 promoters; Strair RK; The adenovirus E1a gene encodes polypeptides which regulate the expression of adenovirus early genes as well as a variety of cellular genes . Although it is likely that the E1a encoded polypeptides regulate the expression of these genes by interaction with a variety of cellular transcription factors, the precise mechanism by which this occurs is currently unknown . This report describes the development of cell lines which contain integrated copies of the E2 promoter driving the expression of the Tn5 neo gene or the Escherichia coli beta-galactosidase gene . In each case phenotypic changes concurrent with expression of the E1a 289 amino acid polypeptide are demonstrated . The use of these cell lines to detect rare events in the activation of the E2 promoter is demonstrated in transfection experiments . These cell lines are also used to study the effects of c-myc expression on integrated E2 promoters.

J Periodontal Res, 1991 Nov, 26(6), 486 - 90
The regulatory effects of monocytes on human natural killer cells activated by lipopolysaccharides; Lindemann RA; Lipopolysaccharides (LPS) rapidly enhance cytotoxicity of human natural killer (NK) cells against tumor targets . The regulatory effects of peripheral blood monocytes (MO) on this activation were measured . When lymphocytes were kept at a constant number in culture containing LPS from oral and enteric bacteria, increasing the percentage of MO caused a dose-dependent suppression of NK cytotoxicity . This suppression was reversed by adding the prostaglandin (PG) inhibitor indomethacin which indicates that PGE was released by MO stimulated by LPS . PGE is known to suppress NK activity by its effects on cAMP . MO separated from lymphocytes by transwell membranes also suppressed NK cells in the presence of LPS but this action was again reversed by indomethacin . This suggests that cell-to-cell contact is not necessary for MO to suppress NK cytotoxicity when stimulated by LPS . The role of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in NK suppression was studied . Antibodies to IL-1 and TNF did not alter the suppression mediated by MO on NK activity . Adding IL-1 or TNF to cell cultures without MO or LPS had no effect on NK activity after 24 h . TNF, but not IL-1, enhanced NK activity in the presence of LPS in cultures without MO . When PGE was preincubated with only lymphocytes for 2 h, the activating effects of a secondary stimulation, interleukin-2 (IL-2), were inhibited . IL-1 had no effect on IL-2 activation when pre-incubated with PBL but TNF slightly enhanced IL-2-induced NK cytotoxicity.

Naturwissenschaften, 1991 Nov, 78(11), 497 - 504
{Biology and biochemistry of selenium}; Forchhammer K et al.; The importance of selenium as an essential trace element has progressively emerged during the last years due to the analysis of selenium deficiency diseases and to the identification and characterization of a number of selenoenzymes . Selenium is incorporated in the catalytic site of the enzymes as an integral selenocysteine residue . The pathway of selenocysteine biosynthesis and incorporation has been elucidated recently for Escherichia coli . This article presents an overview on these subjects and describes the mechanisms which confer selenocysteine specificity in the framework of protein biosynthesis . In addition, some considerations concerning the phylogeny of selenocysteine incorporation are presented and a model for the evolution of the selenocysteine pathway is proposed.

Genetics, 1991 Nov, 129(3), 611 - 21
Chain bias in Chi-stimulated heteroduplex patches in the lambda ren gene is determined by the orientation of lambda cos; Hagemann AT et al.; Heteroduplex patch recombinants have received information in one DNA chain but have not recombined flanking markers . Evidence regarding which chain is exchanged bears on the structure of recombination intermediates . The direction of travel along DNA of RecBCD recombinase, the central enzyme in the Escherichia coli RecBCD pathway of homologous recombination, is determined in phage lambda by the orientation of the packaging origin, cos . cos is a double-chain cut site which serves as a preferred entry site for RecBCD . Using partially denaturing gels to resolve heteroduplex molecules, we have examined patch recombinants at the lambda ren gene . We report that the transferred information in Chi-stimulated patches at ren can occur on either chain, but is biased to the chain ending 5' at the right of the lambda map (the lambda r chain) in phage carrying cos in its normal orientation . The chain bias switches in favor of the chain that ends 3' at the right (the lambda l chain) when RecBCD travel direction is reversed by inverting cos . We entertain models that accommodate these and other results pertaining to the structure of RecBCD-mediated recombinants.

Mol Gen Genet, 1991 Nov, 230(1-2), 295 - 301
DNA sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ and urvA cells; Sockett H et al.; Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E . coli uvr+ and excision-deficient uvrA cells . Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr+ cells . This conclusion is confirmed by analysis of published results for genes in both uvr+ and uvr- cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E . coli lacI gene and of the lambda phage cI repressor gene . Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E . coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair . Otherwise, mutagenesis in hamster cells appeared similar to that in E . coli uvr+ cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells . Phenotypes of 6-thioguanine-resistant E . coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria.

Mol Gen Genet, 1991 Nov, 230(1-2), 170 - 6
The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts; Maeser S et al.; A mutant Gin recombinase of the phage Mu DNA inversion system was successfully expressed in Arabidopsis thaliana and tobacco protoplasts . Site-specific recombination was monitored both physically and biologically with the help of a recombination assay system in which expression of a beta-glucuronidase (gus) gene requires Gin-mediated recombination . We demonstrate that the wild-type Gin protein is not able to promote recombination in plant protoplasts, presumably because plant cells do not contain a protein that can substitute for the Escherichia coli FIS protein needed for full activity of wild-type Gin in E . coli . A FIS-independent Gin mutant protein on the other hand was efficient in promoting recombination on recombination substrates introduced transiently and on substrates stably integrated into the plant genome . We discuss the various advantages this system can provide for genetic manipulation of plant cells.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9513 - 7
DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity; McCarty JS et al.; DnaK, the sole Escherichia coli member of the highly conserved 70-kDa heat shock protein (HSP70) family of proteins, autophosphorylates when incubated with ATP in vitro . We show that threonine-199 is the amino acid that becomes phosphorylated and we demonstrate that threonine-199 is critical for the ATPase activity of DnaK . We also report that both the ATPase and autophosphorylating activities of DnaK increase very strongly over the range of temperatures that is physiologically relevant for E . coli growth . The temperature dependence of either or both of these activities could be of significance with respect to the postulated role of DnaK as a molecular chaperone in helping cells ameliorate the deleterious consequences of elevated temperature . Furthermore, we postulate that DnaK plays a key role in regulation of the heat shock response by serving as a cellular thermometer that directly senses the environmental temperature.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9508 - 12
Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine; Wu J et al.; Mitogen-activated protein kinase (MAP kinase) is a serine/threonine protein kinase that becomes enzymatically activated and phosphorylated on tyrosine and threonine following treatment of quiescent cells with a variety of stimulatory agonists . Phosphorylation on both tyrosine and threonine is necessary to maintain full activity, and these two regulatory phosphorylations occur close to each other, separated by a single glutamate . To study the mechanisms by which MAP kinase becomes phosphorylated and activated, we have cloned a full-length cDNA encoding MAP kinase and have expressed the enzyme in Escherichia coli as a soluble nonfusion protein . We find that the enzyme displays a basal, intramolecular autophosphorylation on tyrosine-185 that is accompanied by activation of the enzyme's kinase activity towards an exogenous substrate . The tyrosine-phosphorylated protein displays a small fraction of the activity seen with the fully activated, doubly phosphorylated enzyme isolated from mammalian cells but is activated 10- to 20-fold relative to the unphosphorylated enzyme . These findings raise the possibility that regulation of MAP kinase activity in response to agonist stimulation could occur in part through the enhancement of autophosphorylation on tyrosine.

J Bacteriol, 1991 Nov, 173(22), 7092 - 7
The first gene in the Escherichia coli secA operon, gene X, encodes a nonessential secretory protein; Rajapandi T et al.; TnphoA insertions in the first gene of the Escherichia coli secA operon, gene X, were isolated and analyzed . Studies of the Gene X-PhoA fusion proteins showed that gene X encodes a secretory protein, since the fusion proteins possessed normal alkaline phosphatase activity and a substantial portion of this activity was found in the periplasm . In addition, the Gene X-PhoA fusion proteins were initially synthesized with a cleavable signal peptide . A gene X::TnphoA insertion was used to construct a strain containing a disrupted chromosomal copy of gene X . Analysis of this strain indicated that gene X is nonessential for cell growth and viability and does not appear to play an essential role in the process of protein export.

Eur J Immunol, 1991 Nov, 21(11), 2775 - 80
Cloning, heterologous expression and characterization of murine interleukin 1 receptor antagonist protein; Shuck ME et al.; A cDNA coding for the human interleukin 1 receptor antagonist protein (IL 1Ra) was used to clone the corresponding murine cDNA . The nucleotide sequence of the open reading frame coding for the processed form of mIL 1Ra predicted a 152-residue protein that was 77% identical to human IL 1Ra . The cellular and tissue distribution of murine IL 1Ra (mIL 1Ra) transcripts showed high levels in macrophages and skin while lower levels were detected in tissues that contain significant numbers of resident macrophages . The portion of the mIL 1Ra cDNA that codes for the mature form of the protein was placed under the control of a Trp promoter and expressed in E . coli at a level of 37% of total cell protein . The expressed protein was secreted into the periplasm and was purified to homogeneity in a single step by cation-exchange chromatography . Recombinant mIL 1Ra competitively inhibited 125I-labeled IL 1 alpha binding to murine type I IL 1R present on EL4 6.1 cells (Ki value of 0.21 nM) and antagonized IL 1-stimulated co-mitogenesis in murine thymocytes (0.7 x 10(6)-1.1 x 10(6) units/mg).

J Neurochem, 1991 Nov, 57(5), 1630 - 5
Modulation of quinolinic and kynurenic acid content in the rat brain: effects of endotoxins and nicotinylalanine; Moroni F et al.; Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring . They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC . The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids . Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid . The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.

Zhongguo Yao Li Xue Bao, 1991 Nov, 12(6), 483 - 8
Influences of Polyactin A on activities of human monocytes in vitro; Hu QL et al.; Effects of Polyactin A (PAA) on abilities of human monocytes to synthesize and secrete interleukin-1 (IL-1) and to modulate natural killer (NK) cell activity in large granular lymphocytes (LGL) were investigated in vitro . Over a wide range of concentrations (0.01-100 micrograms.ml-1), PAA directly induced IL-1 synthesis and secretion, showing the maximal effect at 10 micrograms.ml-1, and evidently synergized with lipopolysaccharides (LPS) of E coli in stimulation of IL-1 production by human monocytes . The manifestation of PAA pretreated autologous monocytes in modulation of NK cell activity was closely related to PAA concentration . A boosting effect of PAA-treated monocytes on NK activity was observed when PAA 10-100 micrograms.ml-1 were used for pretreatment of monocytes, while an inhibitory influence of monocytes was found when PAA 0.01-0.1 microgram.ml-1 were used . These results demonstrate significant effects of PAA on functions of human monocytes, enhancing IL-1 production and affecting their regulative activity on NK cell cytotoxicity.

Cereb Cortex, 1991 Nov-Dec, 1(6), 463 - 8
Separate progenitor cells give rise to pyramidal and nonpyramidal neurons in the rat telencephalon; Parnavelas JG et al.; Neurons of the mammalian cerebral cortex are commonly subdivided into two broad classes: pyramidal and nonpyramidal . The former are projection neurons, while the latter are interneurons . To determine whether the two neuronal classes in the cerebral cortex are derived from the same or separate progenitor cells, we used a recombinant retrovirus containing the reporter gene E-coli beta-galactosidase as a lineage marker . Clonally related neurons expressing the inherited beta-galactosidase gene were detected histochemically, at both light and electron microscopic levels, and their phenotypes were identified using well-established ultrastructural criteria . The clones examined, with one exception, were composed of either all pyramidal or all nonpyramidal neurons . These findings suggest that pyramidal and nonpyramidal neurons in the cerebral cortex have separate lineages and are derived from different progenitor cells in the ventricular zone . This lends weight to the notion that cells in the ventricular zone comprise a heterogeneous population, and that lineage contributes substantially to the phenotype of a neuron.

Glycobiology, 1991 Nov, 1(5), 537 - 42
A method for identifying a proposed carbohydrate-binding motif of proteins; Baumann MA et al.; An examination of the binding sites of four carbohydrate binding proteins (Escherichia coli lactose repressor, E . coli arabinose-binding protein, yeast hexokinase A and Concanavalin A) revealed certain similarities of amino acid sequences and residues forming hydrogen bonds and hydrophobic interactions with the bound carbohydrate . These were: (i) Asx-Asx, hydrogen bonding to the pyranose ring oxygen and anomeric-OH group; (ii) Arg-X-X-X-(Ser/Thr), or the reverse sequence, with the Arg hydrogen bonding to the pyranose ring oxygen; (iii) Lys-(Ser/Thr)-X-X-Asp, or the reverse sequence and with interchange of the Lys-(Ser/Thr) positions, with hydrogen bonding of either or both the Lys and Asp residues to the -OH groups at carbons 2, 3, 4 or 6; (iv) a diaromatic sequence with possible hydrophobic interactions to the faces of the pyranose ring structure . An algorithm was devised to search the amino acid sequences of a large number of proteins, those known to bind carbohydrates as well as those without known carbohydrate-binding activities, for the four amino acid sequence criteria . The algorithm incorporated a weighted distance value (WDV) to assess the approximate distance between any two criteria, with the WDV being based on the predicted secondary structure of the protein amino acid sequence . When the algorithm using criteria 1 and 2 plus the WDV was applied to the sequences of 125 proteins, the method indicated the presence of the potential carbohydrate-binding site motif for 42% of proteins with known carbohydrate binding, only 8% of proteins were predicted as false positives, and the accuracy of the method was calculated to be 61.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Z Naturforsch {C}, 1991 Nov-Dec, 46(11-12), 1063 - 6
{Replication of ColE 1-related plasmids at increased growth temperature depending on rom function}; Riethdorf S et al.; The content of ColE 1-related plasmids increased about 4-6-fold after a temperature-shift from 30 to 42 degrees C (45 degrees C) if the rom-region of the plasmids was deleted . The copy number of rom(+)-plasmids did not change after the temperature shift . All rom(-)-plasmids tested in this study showed this plasmid amplification . The Rom-protein is capable of inhibiting plasmid replication by stabilization the initial reversible stage of the association of RNA I with the primer precursor RNA II . We suggest that the temperature-dependent enhancement of the copy number of rom(-)-plasmids is due to a destabilization of this initial phase of the RNA I-preprimer interaction at high temperatures which is suppressed by the Rom-protein in cells with rom(+)-plasmids.

Cell Growth Differ, 1991 Nov, 2(11), 561 - 6
Production and functional characterization of human recombinant FGF-6 protein; Pizette S et al.; The fibroblast growth factor (FGF) gene family to date comprises seven members and has been implicated in a wide range of physiological and biological processes, including angiogenesis, morphogenesis, and tumorigenesis . The FGFs are mitogens for a broad range of cells of various embryological origins and can act as differentiation factors . The FGFs can bind to tyrosine kinase and non-tyrosine kinase transmembrane receptors; the physiological basis for this is still unknown . In order to study more thoroughly the activities of FGF-6, we have constructed a bacterial expression vector by inserting FGF-6 complementary DNA sequences into the T7 RNA polymerase-based pET3a vector . The resulting construct is able to drive the expression of a high amount of FGF-6 protein in Escherichia coli, which can be solubilized and purified through heparin-Sepharose chromatography and high salt elution . The purified FGF-6 protein displays a strong mitogenic activity on BALB/c 3T3 cells and is able to morphologically transform these cells . By contrast, adult bovine aortic endothelial cells, which normally require the presence of FGF-2 for their growth, show only a limited mitogenic response that is highly dependent on heparin concentration.

Mol Biol (Mosk), 1991 Nov-Dec, 25(6), 1533 - 8
{Structure-activity study of the HindIII-I fragment of the L-IVP strain of vaccinia virus genome . II . Cloning the I(sub 6) gene and identification of its protein product}; Chikaev NA et al.; The I6 gene from the HindIII-I-fragment of the vaccinia virus strain L-IVP DNA was cloned into bacterial vector pUC19 . The monospecific antiserum to the expression product of this gene in E . coli was obtained . Using this antiserum the I6 gene was shown to code the viral protein of 34 kDa molecular weight estimated from SDS-PAGE . This protein is not included into the mature virion, but can be detected in the cytoplasm of the vaccinia virus infected cells.

Mol Biol (Mosk), 1991 Nov-Dec, 25(6), 1526 - 32
{A structure-activity study of the HindIII-I fragment of the L-IVP strain of vaccinia virus genome . I . Cloning of T5 gene and identification of its protein product}; Netesova NA et al.; The I5 gene from the HindIII-I-fragment of the vaccinia virus strain L-IVP DNA was cloned into bacterial vector pUC19 . The monospecific antiserum to the expression product of this gene in E . coli was obtained . This antiserum was demonstrated to co-precipitate the virion protein p90 . The vaccinia virus strain L-LVP DNA was shown to have only one ORF coding the p90 protein instead of two ORF H5 and H4 as known for vaccinia virus strain WR . This protein is associated with the core of vaccinia virion, but some of its antigenic determinants are exposed on the surface of the viral particles.

J Endod, 1991 Nov, 17(11), 561 - 6
In vitro evaluation of an hydroxyapatite root canal system filling material; White JM et al.; An objective of endodontics is the three-dimensional filling of the root canal system, preventing leakage . The sealing ability of two hydroxyapatite formulations was compared with that of a gutta-percha/sealer combination placed with a vertical compaction technique . Zones of lysis tests of the hydroxyapatites showed no bacteriocidal effect of the formulations . Bacterial challenges of the sealing abilities of the formulations and gutta-percha/sealer combination showed that the formulations were not superior to the gutta-percha/sealer combination in their apical seal.

Biofizika, 1991 Nov-Dec, 36(6), 1069 - 78
{Distribution of carbon isotopes ((13)C/(12)C) in cells and temporal organization of cellular processes}; Ivlev AA; Recent studies on fractionation of carbon isotopes in biological systems are reviewed . It follows that direct experimental proofs have been obtained that 1) basic fractionation of carbon isotopes in the cell is related to isotope effect in pyruvate decarboxylation; 2) fractionation of carbon isotopes in the above reaction in vivo proceeds with exhausting substrate pool . The latter provides natural relationship between metabolites isotope distribution and sequence of their synthesis in the cell cycle, or with the temporal organization of cellular metabolism . The non-steady and periodic pattern of pyruvate decarboxylation due to the exhausting substrate pool well agrees with the existing notions on reciprocal oscillations in the cell glycolytic chain . Experimental data are presented corroborating indirectly the existence of oscillations in bacterial cells . Earlier proposed model of the mechanism of carbon isotope fractionation based on the above principles can be used for analysing changes in isotopic characteristics of the organisms and interpreting their relations with metabolic processes.

Curr Genet, 1991 Nov, 20(5), 385 - 9
Altered expression of the steroid bioconverting pathway in pAN 7-1 transformants of Cochliobolus lunatus; Rozman D et al.; The filamentous fungus C . lunatus converts progesterone mainly to its 11 beta-hydroxy derivative . C . lunatus transformed with the plasmid pAN 7-1, which contains the E . coli hph gene expressed under the control of the A . nidulans gpd and trpC expression signals, lacks this activity, but exhibits acetyl side chain degradation of progesterone through the reaction scheme progesterone----20 beta-hydroxy-progesterone----delta 4-androstene-3,17-dione---- testolactone + testosterone . The main part of this metabolic pathway is not expressed in the non-transformed strain . It was determined that the site-specific integration of the plasmid into the genome directly influences the expression of genes involved in the bioconversion of steroids.

Biotechniques, 1991 Nov, 11(5), 648 - 9
Rapid purification of a recombinant protein using tandem radial flow ion-exchange column chromatography; McCartney JE; Tandem radial flow anion- and cation-exchange columns were used to partially purify and concentrate a dilute recombinant protein that had been refolded in vitro after production as insoluble inclusion bodies in E . coli . The refolded sample was first passed through a Q-Sepharose Fast Flow column in order to remove the majority of E . coli contaminating proteins and endotoxins, then purified on an S-Sepharose Fast Flow column connected to the outlet of the Q-Sepharose column . This tandem arrangement enabled the rapid processing of multiple preparations of refolded material during production method development.

Antimicrob Agents Chemother, 1991 Nov, 35(11), 2444 - 6
Inhibitory effects of recombinant human cystatin C on human coronaviruses; Collins AR et al.; Cystatin C, a potent inhibitor of cysteine proteases such as papain and cathepsin B, was examined for its effect on human coronaviruses OC43 and 229e . Both viruses were greater than 99% inhibited by 0.1 mM inhibitor . Endpoint titrations showed that inhibiting activity paralleled that of leupeptin, a serine and cysteine protease inhibitor, and indicated that 1 to 2 microM inhibitor, slightly above physiologic levels, was effective.

Biochimie, 1991 Nov, 73(11), 1391 - 6
The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU; Spinelli S et al.; The crystal structure of the aspartyl protease encoded by the gene pol of the human immunodeficiency virus (HIV-1, isolate BRU) has been determined to 2.7 A resolution . The enzyme, expressed as an insoluble denatured polypeptide in inclusion bodies of Escherichia coli has been renatured and crystallized . It differs by several amino acid replacements from the homologous enzymes of other HIV-1 isolates . A superposition of the C alpha-backbone of the BRU protease with that of the SF2 protease gives a roots mean square positional difference of 0.45 A . Thus, neither the denaturation/renaturation process nor the amino acid replacements have a noticeable effect on the three-dimensional structure of the BRU protease or on the detailed conformation of the catalytic site, which is very similar to that of other aspartyl proteases.

Biochimie, 1991 Nov, 73(11), 1387 - 9
Overexpression of L7/L12 protein with mutations in its flexible region; Gudkov AT et al.; Using restriction enzymes and polymerase chain reaction, three mutated forms of L7/L12 proteins with deleted 38-46, 44-52 and 38-52 residues were obtained . These mutant proteins were isolated in a preparative scale and were shown to bind to ribosomes and to affect ribosomal function.

Anal Biochem, 1991 Nov 1, 198(2), 292 - 7
Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate; Minobe S et al.; The LAL test is inhibited or enhanced by many substances . To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins) . This method is composed of two steps . The first step is the adsorption of endotoxins . Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit . The second step is the reaction of adsorbed endotoxins with the LAL reagent . The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay . The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95% . The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.

Pharm Res, 1991 Nov, 8(11), 1384 - 8
113Cd nuclear magnetic resonance (NMR) study of the inhibitory effect of methylvinylether/maleic acid (PVM/MA) copolymer on the alkaline phosphatase of Escherichia coli; Afflitto J et al.; The inhibitory effect of PVM/MA copolymer on the alkaline phosphatase (AP) of E . coli was investigated . Kinetic studies indicated that enzyme inhibition was characterized by a reduction in both the Vmax and the Km . Addition of 1 mM zinc or magnesium ions to the reaction prevented inhibition of the enzyme by the copolymer . The inhibitory effect of the copolymer on alkaline phosphatase was also investigated using 113Cd NMR after exchange of the active center metal ions with 113Cd . The resulting Cd(II)6AP exhibited characteristic 113Cd resonances reflecting the environment of the A, B, and C metal binding sites of the enzyme's active center . Addition of copolymer resulted in a 113Cd NMR spectrum which indicated removal of 113Cd from the C site and formation of two distinct forms of the enzyme . Possible explanations for the 113Cd NMR results are discussed.

Br J Pharmacol, 1991 Nov, 104(3), 691 - 9
Sequential release of tumour necrosis factor, platelet activating factor and eicosanoids during endotoxin shock in anaesthetized pigs: protective effects of indomethacin; Mozes T et al.; 1 . The effects of indomethacin were investigated on haemodynamics, haematological and blood glucose values, and the release of tumour necrosis factor (TNF), platelet activating factor (PAF) and eicosanoids in anaesthetized pigs receiving 5 micrograms kg-1 E . coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery . The animals were observed for an additional period of 2 h after the termination of LPS infusion . 2 . Eight of the 17 animals infused with LPS and not treated with indomethacin died within 30 min after the beginning of LPS infusion (non-survivors), while the other 9 survived the experimental period of 3 h though in a state of shock (survivors) . 3 . No alterations were observed in plasma concentrations of PAF and eicosanoids (thromboxane B2 (TXB2), 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and leukotriene B4 (LTB4} in non-survivors . However, a marked increase was detected in TNF release . A significant, though transient, increase in concentrations of PAF, TNF and eicosanoids occurred in the survivors . The peak in the concentrations of PAF and TXB2 preceded the maximum in TNF values in survivors . 4 . Another group of 7 LPS-infused pigs was treated with indomethacin (2 mg kg-1, i.v . bolus 60 min before the start of LPS infusion, followed by a continuous infusion of 3 mg kg-1 h-1) . This treatment prevented death and shock despite the high concentrations of circulating TNF and PAF . Concentrations of cyclo-oxygenase enzyme products were reduced, whereas LTB4 release was not affected . The effect of indomethacin on haemodynamic changes occurred earlier than on cyclo-oxygenase products.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, 1991 Nov, 12(11), 955 - 94
The gene-protein database of Escherichia coli: edition 4; VanBogelen RA et al.; The gene-protein database of Escherichia coli has as its core an index that links each of the protein spots from a two-dimensional polyacrylamide gel to the gene that encodes the protein . Additional information about each protein and its gene is generated from two-dimensional gel analysis or collated from the literature to form the database . Earlier editions of the database have provided periodic updates of information . The current edition does this, but also introduces a new reference gel image produced by an electrophoresis system recently adopted in this laboratory . The new gel system was chosen because it offers an improved opportunity for other investigations to produce close replicas of the reference gel pattern, thereby allowing easier access to the information of the database and encouraging independent contribution to the database . The new gel format also is larger and hence more compatible with computer assisted image analysis, which has become essential for a project of this magnitude . This edition continues the use of the former reference gel images, but adds a reference image of an equilibrium gel of E . coli strain W3110 produced by the new standardized gel system . At this time, 55% of the protein spots annotated on the previous equilibrium reference gel for this organism have been located on the new reference image, and these identifications are included in the tables of the database.

J Reprod Fertil, 1991 Nov, 93(2), 525 - 39
Identification of molecules involved in the 'early pregnancy factor' phenomenon; Clarke FM et al.; An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties . N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA . The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified . Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence . Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs . Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e . to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs . Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.

Appl Environ Microbiol, 1991 Nov, 57(11), 3062 - 9
Isolation of a neuraminidase gene from Actinomyces viscosus T14V; Yeung MK et al.; A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities . Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid . Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region . Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector . Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da . The protein from cell extracts of E . coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel . Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E . coli were hemagglutinated by Actinomyces spp . The enzyme expressed by E . coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues . Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A . viscosus T14V . The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A . viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases . Data from hybridization studies show that A . viscosus T14V contains a single copy of the neuraminidase gene.

Res Vet Sci, 1991 Nov, 51(3), 272 - 7
Lactating cows become partially refractory to frequent intramammary endotoxin infusions: recovery of milk yield despite a persistently high somatic cell count; Shuster DE et al.; Midlactation cows were infused with 10 micrograms endotoxin in the same two homolateral quarters after each of several consecutive milkings to study the effect of prolonged, endotoxin-induced mastitis on lactational performance . The initial infusion induced an acute response with systemic involvement . Inflammation developed in infused quarters, and milk production declined and milk composition was altered in all quarters . Subsequent infusions failed to induce systemic responses . Furthermore, milk yield and composition in uninfused quarters returned to pre-treatment levels despite further infusions . In infused quarters, milk yield, protein percentage and serum albumin concentration showed partial recovery during the endotoxin infusion period . In contrast, decreased lactose concentration, and increased cell count, N-acetyl-beta-D-glucosaminidase and lactoferrin levels persisted throughout the infusion period . After infusions were stopped, all measurements returned to near pretreatment levels . These data demonstrate that systemic, but not local, responses become refractory to multiple intramammary endotoxin infusions, and that multiple infusions have continued but little progressive or permanent, inhibitory effects on lactational performance despite a persistent mammary leucocytosis.

Mol Microbiol, 1991 Nov, 5(11), 2807 - 14
Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon; Rossmann R et al.; The products of a minimum of 15 genes are required for the synthesis of an active formate-hydrogenlyase (FHL) system in Escherichia coli . All are co-ordinately regulated in response to variations in the oxygen and nitrate concentration and the pH of the culture medium . Formate is obligately required for transcriptional activation of these genes . Analysis of the transcription of one of these genes, hycB linked to the lacZ reporter gene, revealed that oxygen and nitrate repression of transcription could be relieved completely, or partially in the case of nitrate, either by the addition of formate to the medium or by increasing the copy number of the gene encoding the transcriptional activator (fhlA) of this regulon . These studies uncovered a further level of regulation in which the transcription of hycB was reduced in cells grown on glucose . This effect was most clearly seen in aerobically grown cells when formate was added externally . Addition of cAMP overcame this glucose repression, which could be shown to be mediated by the cAMP receptor protein . These results would be consistent with the transport of formate being regulated by catabolite repression . Moreover, the repression of transcription through high pH also could be partially overcome by addition of increasing concentrations of formate to the medium, again being consistent with regulation at the level of formate import and export . Taken together, all these observations indicate that it is the intracellular level of formate that determines the transcription of the genes of the formate regulon by FhlA . This represents a novel positive feedback mechanism in which the activator of a regulon induces its own synthesis in response to increases in the concentration of the catabolic substrate, and this in turn is governed by the relative affinities of FhlA and the three formate dehydrogenase isoenzymes for formate.

Mol Microbiol, 1991 Nov, 5(11), 2727 - 33
Investigation of the specificity of the interaction between colicin E9 and its immunity protein by site-directed mutagenesis; Curtis MD et al.; Comparison of the amino acid sequences of the C-terminal domain of three DNAase type E colicins has identified six candidate specificity determinants for the interaction of these E colicins with their homologous immunity proteins . We have changed these candidate specificity determinants of colicin E9, using site-directed mutagenesis, to the corresponding amino-acids of colicin E8 . A 'mutant' colicin E9, in which four of the six candidate specificity determinants have been changed, demonstrated colicin activity against Escherichia coli indicator strains which carried either the E8imm or the E9imm genes, indicative of a 'novel' E . colicin . After changing all six of the candidate specificity determinants, the resulting colicin E9 'mutant' exhibited a phenotype very similar to that of colicin E8.

Mol Microbiol, 1991 Nov, 5(11), 2719 - 25
Escherichia coli rpoA mutation which impairs transcription of positively regulated systems; Thomas MS et al.; The rpoA341 (phs) mutation of Escherichia coli results in decreased expression of several positively regulated operons and has been mapped to within or very near the rpoA gene encoding the alpha subunit of RNA polymerase . We have shown that plasmid-directed synthesis of the wild-type alpha subunit can complement the defective phenotypes associated with this mutation consistent with its proposed location within rpoA . This mutation was mapped by marker rescue to within a 182bp region near the 3' end of rpoA and was subsequently transferred to a plasmid by recombination in vivo . DNA sequence analysis revealed that the RpoA341 phenotype was the result of the substitution of lysine 271 by glutamate within the alpha polypeptide . We discuss this result in relation to our current understanding of the functional organization of the alpha subunit.

Mol Microbiol, 1991 Nov, 5(11), 2685 - 93
The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co-ordinated action of the Kis and Kid proteins; Ruiz-Echevarria MJ et al.; Stability mediated by the parD system of plasmid R1 is modulated by a killer protein, Kid, and by an antagonist of this function, Kis . Determination of the 5' ends of ParD transcripts, revealed that the genes coding for these proteins are transcribed from a single promoter . Analysis of the 3' end of the ParD RNAs indicated the existence of two transcripts: one of them coding for the Kis and Kid proteins, and the other coding only for Kis . Analysis of the effects of parD+ and kis+ recombinants on the beta-galactosidase levels expressed by different transcriptional and translational parD-lacZ fusions, and on the ParD RNA levels determined by a derepressed parD mutant, indicated that the Kis and Kid proteins repress coordinately the parD system at the transcriptional level . We discuss the relevance of these results in terms of the activities of the Kis and Kid proteins and in the context of the stabilization mediated by parD.

Mol Microbiol, 1991 Nov, 5(11), 2629 - 40
Escherichia coli molecular genetic map (1500 kbp): update II; Medigue C et al.; The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al . (1987) and modified by others . This second update adds a further 500 kbp, increasing the amount of the E . coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base . The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene . The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file . The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences . There appears to be a significant counter-selection against several such sequences in E . coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E . coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.

Mol Microbiol, 1991 Nov, 5(11), 2599 - 610
Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function; Fee JA; This review is concerned with the effects of environmental perturbations on the expression of the two superoxide dismutase (SOD) genes in Escherichia coli (sodA, MnSOD; sodB, FeSOD) . Early studies using SOD activity, showed that MnSOD levels respond to changes in oxygen tension, type of substrate, redox active compounds, iron concentration, the nature of the terminal oxidant, and the redox potential of the medium . FeSOD levels appeared nominally insensitive to these perturbations . More recent molecular genetic studies revealed that sodA expression is subject to regulation by three major regulatory systems: fur (ferric uptake regulation) and arcA arcB (aerobic respiratory control) mediate repression of sodA, while a relatively new system, soxR soxS (superoxide response), mediates activation of sodA expression . By contrast, sodB expression, which is much less studied at this time, appears to be positively activated in trans by fur . A rudimentary gene regulation model is presented which rationalizes past observations, is experimentally testable, and should serve as a guide to future research in this area.

J Pediatr Gastroenterol Nutr, 1991 Nov, 13(4), 409 - 14
Persistent diarrhea: total gut transit time and its relationship with nutrient absorption and clinical response; Roy SK et al.; The study was undertaken to better understand the role of total gut transit time (TGTT) on the absorption of nutrients in patients with persistent diarrhea . Twenty-six boys aged 3-18 months with persistent diarrhea and 25 age-matched healthy controls were studied . Their TGTT was measured with charcoal markers during their treatment with a diet made up with rice powder soya oil, glucose, and egg white . Coefficients of absorption of nutrients were estimated in a 72-h balance study . The median TGTTs in patients and controls were 5 and 11.6 h, respectively . Among the patients, the TGT correlated significantly with absorption of total energy (p less than 0.01), absorption of fat (p less than 0.01), stool frequency (p less than 0.01), and stool weight during the 1st 24 h (p less than 0.01) . Coefficients of absorption of energy, fat, and carbohydrate were significantly different among the patients above or below the median transit time (5 h) . None of these relationships was present among the healthy controls . The TGTT was negatively associated with the duration of clinical recovery . The results of this study suggested that intestinal transit time is an important factor for absorption of nutrients that may influence clinical recovery in patients with persistent diarrhea.

J Pediatr Gastroenterol Nutr, 1991 Nov, 13(4), 402 - 8
Infectious gastroenteritis does not act as a triggering mechanism for the synthesis of serum IgG antibody to beta-lactoglobulin; Brussow H et al.; beta-Lactoglobulin (BLG)-specific serum IgG antibody was measured by enzyme-linked immunosorbent assay in 1,392 serum samples from newborn to 5-year-old Ecuadorian children enrolled into a representative nutrition and health survey . At a 1:100 serum dilution, 62% of the children showed specific antibody (blank-corrected optical density greater than or equal to 0.1) . This prevalence did not change with increasing age . More specifically, we did not observe a prevalence or titer increase of BLG-specific antibody in age groups where the majority of these Ecuadorian children experienced infection with rotavirus (8-24-month age groups) and enterotoxigenic Escherichia coli (8-12-month age group) . In addition, BLG-specific antibody did not differ between children who did or did not experience an episode of diarrhea 15 days before blood sampling . We observed a small but statistically significant difference in BLG-specific antibody between subsamples of Ecuadorian children regularly or only occasionally ingesting milk . Titers were higher in the group consuming more milk.

Circ Shock, 1991 Nov, 35(3), 164 - 73
Renal function and metabolism during endotoxemia in rats: role of hypoperfusion; van Lambalgen AA et al.; Renal failure often complicates endotoxin shock . This might be due to renal hypoperfusion, but endotoxemia could also have additional effects . We studied in anesthetized rats renal plasma flow (RPF), glomerular filtration rate (GFR), and metabolism (ATP, CrP = creatine phosphate, energy charge = {ATP + 0.5 ADP}/{ATP + ADP + AMP}, lactate, glucose) during endotoxin shock (Escherichia coli endotoxin, 10 mg/kg for 60 min; n = 10) and "balloon shock" (balloon inflated in vena cava below renal vein to cause comparable decreases in cardiac output and RPF as in endotoxin-treated rats; n = 10) . A third group of rats served as controls (n = 10) . At t = 0 infusion of endotoxin was started . At t = 90 min, when cardiac output was low and serum lactate was high (indicating shock), GFR and RPF were obtained from plasma disappearance rates (from t = 90 to t = 135 min) of 125I-thalamate and 131I-hippurate, respectively . Experiments ended at t = 135 min . In both shock groups RPF decreased (by ca . - 75% compared with control rats), but filtration fraction only increased (by 72%) in the "balloon shock" rats . In renal biopsies lactate concentration increased more (by 407 vs . 167%) and ATP decreased more (by -63 vs . - 35%) during endotoxin shock than during "balloon shock"; the endotoxin-treated rats also showed a significant decrease in CrP (by - 58%), energy charge (by - 31%), and glucose concentration (by - 34%), and an increase in the number of leukocytes in the glomeruli (by 730%) . Renal function and metabolism thus was more affected in this hypodynamic form of endotoxin shock than in "balloon shock." This may be caused by the effects of endotoxin on sticking of leukocytes and renal metabolism.

New Biol, 1991 Nov, 3(11), 1074 - 88
Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals; Zhao LJ et al.; Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41 . These clusters are designated BS I, BS II, and BS III, respectively . (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal) . The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible . However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E . coli beta-gal fusion protein . Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins . Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.

Neurochirurgia (Stuttg), 1991 Nov, 34(6), 188 - 90
A wooden foreign body penetrating the superior orbital fissure; Zentner J et al.; The case of a 12-year-old patient with a wooden foreign body which had penetrated the superior orbital fissure is presented . Using a transethmoidal approach, only some splinters lying in the periorbital soft tissue were removed . The patient became febrile, indicating an infectious complication due to a retained foreign body . This was confirmed by CT scan and MRI demonstrating a main splinter in the superior orbital fissure . Total removal of the wood was achieved via a pterional extradural approach . The difficulties of identifying wooden foreign bodies as well as the topographical problems involved with the approach to the superior orbital fissure are discussed.

J Clin Microbiol, 1991 Nov, 29(11), 2522 - 7
Virulence factors associated with cytotoxic necrotizing factor type two in bovine diarrheic and septicemic strains of Escherichia coli; Oswald E et al.; Forty-three bovine isolates of Escherichia coli producing a second type of cytotoxic necrotizing factor (CNF2) and three K-12 strains carrying different Vir plasmids coding for CNF2 were tested for the presence of several virulence factors . Most of the strains were serum resistant (79%), produced an aerobactin (70%), and adhered to calf villi (53%); some of them produced a colicin (32%) and a hemolysin (9%) . These strains were also tested by a colony hybridization assay with gene probes for six toxins (classical heat-stable {STaP and STb} and heat-labile {LT-I and LT-IIa} enterotoxins and Shiga-like toxins {SLT-I and SLT-II}) and five adhesion factors (K99, K88, 987P, F17, and F41) . Only two gene probes, LT-IIa (9%) and F17A (53%), hybridized with the CNF2 strains . However, antibodies raised against F17 fimbriae did not agglutinate the strains hybridizing with the F17A probe . In contrast, all except one of these strains adhered to calf villi . Interestingly, these two properties, F17A positivity and adherence to calf villi, were the only ones expressed by the K-12 strains carrying different Vir plasmids . In conclusion, this study confirmed that CNF2-producing strains are unrelated to previously described toxigenic E . coli strains and also demonstrated that in half of the strains the production of CNF2 was associated with an adhesion factor genetically related to, but different from, F17, which is more than likely encoded by Vir plasmids.

J Clin Microbiol, 1991 Nov, 29(11), 2418 - 23
110-kilodalton recombinant protein which is immunoreactive with sera from humans, dogs, and horses with Lyme borreliosis; Caputa AC et al.; EcoRI-digested DNA from Borrelia burgdorferi was ligated into the dephosphorylated vector pWR590 and transformed into Escherichia coli DH5 alpha . When the gene library was screened, 20 clones reacted with pooled dog sera with high titers (immunofluorescent antibody titer, greater than or equal to 1,280) to this spirochete . One clone expressed a 110-kDa antigen that reacted strongly with the high-titered pooled sera from dogs with Lyme borreliosis and serum from goats immunized with B . burgdorferi . The 110-kDa protein was serum from goats immunized with B . burgdorferi . The 110-kDa protein was expressed with and without isopropyl-beta-D-thiogalactosidase, indicating the protein is not a fusion protein with beta-galactosidase . Monospecific antisera to the 110-kDa antigen recognized a 75-kDa Borrelia protein . Of the sera that reacted with B . burgdorferi by immunoblotting; 57, 100, and 83% of human, dog, and horse serum samples, respectively, reacted with the 110-kDa protein . Sera from individuals that tested negative with a B . burgdorferi lysate with immunoblotting showed no reaction with the 110-kDa protein . The 110-kDa antigen appears to be useful for the diagnosis of Lyme borreliosis.

J Clin Microbiol, 1991 Nov, 29(11), 2375 - 9
Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimens by an immobilized, colorimetric, nested polymerase chain reaction; Hornes E et al.; A combination of selective enrichment by using immunomagnetic separation of F4 (K88)-positive Escherichia coli and a nested colorimetric polymerase chain reaction (PCR) was used on crude clinical and spiked samples for determination of genes encoding heat-stable enterotoxins (STs) Ia (ST Ia) and Ib (ST Ib) . The combination increased the sensitivity of the nested PCR compared with that of application onto crude samples . Dead cells were also enriched by use of this technology, giving results that are not available by traditional cultivation as enrichment before PCR . The second step in the PCR was modified to be able to differentiate between ST Ia and ST Ib genes . The colorimetric PCR was performed in a microtiter format, making it useful for automation in clinical laboratories and for the screening of large numbers of samples.

Zentralbl Veterinarmed A, 1991 Nov, 38(9), 641 - 51
Bovine endometritis: current and future alternative therapy; Hussain AM et al.; Abnormal parturition can be followed by a persistent endometritis which can have deleterious effects on the cow's subsequent reproductive performance . Normal and active uterine defense mechanisms have been reported to be very important for the exclusion of bacterial infection from the uterus and recovery from endometritis developing after parturition . Despite the widespread use of local or systemic antibiotics, antiseptics, sulfonamides and hormones, rates of recovery from endometritis and the cow's subsequent fertility have not increased appreciably . Furthermore, the cost of any treatment, the frequency of its administration and the milk disposal after treatment make their use uneconomic . Alternative therapies which stimulate the natural uterine defense mechanisms have been suggested as treatments of bovine endometritis . These include: (I) Endotoxins such as lipopolysaccharide of E . coli, (II) serum, plasma or hyperimmune serum, (III) polymorphonuclear leucocyte (PMN) extracts and components and (IV) granulocyte-macrophage colony stimulating factors (G-M CSF).

Vet Microbiol, 1991 Nov, 29(3-4), 299 - 307
Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays; Osek J et al.; We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests . K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method . LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies . It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type . There was 100% correlation between the slide agglutination and ELISA tests . Of the 103 strains tested 68.9% produced LT or STa or both toxins . LT-positive strains were the most common ones in both groups of piglets . All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study . Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E . coli strains isolated from piglets.

Vet Pathol, 1991 Nov, 28(6), 519 - 23
The effects of Escherichia coli and its endotoxin on amyloidogenesis in ducks; Ling YS et al.; To compare the effects of two inocula on the induction of amyloidosis in normal and thymectomized ducks, 180 normal and 50 thymectomized ducks were injected intravenously weekly for up to 16 weeks with either crude endotoxin or crude whole cell extract of a virulent strain of Escherichia coli (O78), and another 60 ducks were injected with normal saline as study control . During the first 5 weekly injections, the initial doses of inducing agents were the smallest and then adjusted upward to the maximum study doses (1 or 2 mg/bird of crude endotoxin and 15-18 x 10(8) bacteria/bird of crude whole cell extract), which were then maintained over the course of the study . The incidence of amyloid deposition were: 50.00% (25/50) for thymectomized ducks that received 1 mg/bird/week of crude endotoxin, 61.67% (37/60) for intact ducks that received 15 x 10(8)-18 x 10(8) bacteria (crude whole cell extract)/bird/week, 53.33% (32/60) for intact ducks received 2 mg/bird/week of crude endotoxin, and 63.33% (38/60) for intact ducks received 1 mg/bird/week of crude endotoxin . The results suggest that crude whole cell extract and crude endotoxin induced amyloidosis in ducks at similar rates and that, in ducks, thymectomy has no appreciable effect on the occurrence of amyloidosis.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 37 - 40
Elimination of broad-host range plasmid vectors in Escherichia coli by curing agents; Bharathi A et al.; A comparative study was made of the susceptibility of broad-host range vector plasmids belonging to Inc P1 and Q groups in Escherichia coli to various curing agents . Plumbagin and SDS eliminated RP4 (Inc P1 group) plasmid whereas pKT231 (Inc Q) and pRK2013 (having ColE1 replicon) were eliminated by hexamine ruthenium (III) chloride, alpha-santonin, coumermycin A1 and cis-dichloro diamine platinum (II) . The curing activity of these agents was specific.

Somat Cell Mol Genet, 1991 Nov, 17(6), 601 - 7
Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes; Grossman M et al.; Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases . One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo . We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures . Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor . The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell . Cytochemical assays were used to detect expression of the recombinant derived proteins, E . coli beta-galactosidase and rabbit LDL receptor . Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels . The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans.

Radiobiologiia, 1991 Nov-Dec, 31(6), 803 - 14
{The phenomenon of adaptive response in radiobiology}; Filippovich IV; Consideration is given to various adaptive reactions to low-level radiation, their association with an absorbed dose, dose rate, radiation quality and time-interval between exposures, as well as with a cell cycle phase . Possible mechanisms of the adaptive response and the character and role of DNA damages, that can induce gene expression of the adaptive response, are discussed . The data on the influence of a preliminary long-term exposure to low-level radiation on the radiosensitivity of biological objects are analyzed with due regard for the adaptive cell response . It is concluded that the adaptive response of cells to ionizing radiation is a particular case of the phenomenon of cell adaptation to the effect of genotoxic factors of the environment.

J Lipid Mediat, 1991 Nov, 4(3), 309 - 25
Platelet activating factor is one of the mediators involved in endotoxic shock in pigs; Mozes T et al.; The role of platelet activating factor (PAF) in endotoxic shock was investigated in anaesthetized pigs receiving 5 micrograms/kg E . coli endotoxin (LPS) into the superior mesenteric artery over a 60 min period . Concentrations of PAF and tumor necrosis factor (TNF) were measured in blood obtained from the superior mesenteric vein and aorta before, during and 60 min after the LPS infusion . The effect of 4 mg/kg of BN 52021, a PAF receptor antagonist, given as a bolus injection 5 min prior to LPS infusion and/or PAF administration into the superior mesenteric vein was studied on systemic and regional hemodynamic variables . Eight of the 17 animals infused with LPS died within 30 min after start of LPS, while the other 9 survived the experimental period of 3 h, though in a shock state . In survivors, PAF concentration in both superior mesenteric vein and aorta increased twenty-fold at 30 min of endotoxaemia, but rapidly returned back towards normal values . No changes in PAF release, but a marked rise in TNF production were measured in non-survivors . Exogenous administration of PAF (0.01 micrograms/kg) produced similar hemodynamic effects as observed in survivors . BN 52021 markedly reduced the effects of PAF on arterial blood pressure for over 1 h . Treatment with BN 52021 (4 mg/kg), injected 5 min prior to LPS infusion, failed to exert any effect on the surviving rate . However, in survivors all circulatory and laboratory parameters studied were improved after treatment with BN 52021 . PAF release observed during LPS infusion in survivors may play a role in the development of shock; however, its role in the rapid death seems to be negligible . Present results clearly demonstrate that endotoxin shock is not crucially dependent on one class of mediators.

Biomaterials, 1991 Nov, 12(9), 853 - 60
Adhesion of Escherichia coli on to a series of poly(methacrylates) differing in charge and hydrophobicity; Harkes G et al.; The adhesion of three Escherichia coli strains on to six poly(methacrylates) differing in hydrophobicity and surface charge was measured as a function of time under laminar flow conditions . Polymers used were poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA) and copolymers of MMA or HEMA with either 15% methacrylic acid (MAA) or 15% trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl) . Bacterial and polymer surfaces were characterized by means of water contact angles and zeta potentials . Both the sessile drop contact angles and the zeta potentials of the bacterial surfaces were significantly different . No significant differences in the sessile drop contact angles of the polymer surfaces were observed . Using the Wilhelmy plate technique large contact angle hysteresis was observed for the different polymer surfaces . Surfaces of copolymers with MAA had more negative zeta potentials than those of the corresponding homopolymers . Surfaces of copolymers with TMAEMA-Cl had positive zeta potentials . The highest numbers of adherent bacteria were found on materials with positive zeta potentials, irrespective of the bacterial strain used . Bacterial adhesion on to copolymers with MAA was less than on to the corresponding homopolymers . Bacterial equilibrium adhesion values correlate with the zeta potentials of the polymer surfaces (r greater than 0.85) . On substrates with less negative zeta potentials high numbers of adhered bacteria were observed . Additionally, the equilibrium bacterial adhesion values could be related with receding contact angles of polymer surfaces with negative zeta potentials (r greater than 0.86) . High equilibrium adhesion values were obtained for polymers with high contact angles . No correlation between the zeta potentials and contact angles of the bacteria with the adhesion values was found.

Acta Anaesthesiol Scand, 1991 Nov, 35(8), 745 - 9
Prevention of endotoxin-induced increase of cerebral oxygen consumption in dogs by propranolol pretreatment; Westerlind A et al.; Cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) were studied in experimental endotoxic shock in dogs . Eight animals were pretreated with a beta-adrenoceptor blocking agent, propranolol (PPL), per os 12 mg/kg once a day for 7 days . Ten animals served as controls . After an intravenous injection of endotoxin, 1 mg/kg, CBF decreased in both groups, with no significant differences between the groups . CMRO2 increased in the control animals by about 18% from the baseline value both 1 and 2 h after the injection of endotoxin . CMRO2 in the PPL-pretreated animals was unchanged after endotoxin . The CMRO2-reactions to endotoxin in control and PPL animals were significantly different after both 1 and 2 h (P less than 0.05) . The present results indicate that the increase in CMRO2 following intravenous endotoxin is mediated via beta-adrenoceptors.

Clin Radiol, 1991 Nov, 44(5), 354 - 6
Case report: hepatobronchial fistula complicating amoebiasis, treated by percutaneous catheter drainage; Stables GI et al.; We report the case of a 41-year-old man who presented with hepatic amoebiasis 18 months after visiting the Far East . His progress was initially complicated by a bronchohepatic fistula which was further complicated by secondary bacterial infection . The fistula persisted and only resolved following percutaneous catheter drainage, thereby avoiding open surgery.

Br J Surg, 1991 Nov, 78(11), 1329 - 31
Effect of biliary infection on common bile duct healing in the rat; Rozga J et al.; It has been postulated that biliary infection plays a role in bile duct stricture formation . The aim of this study was to verify this hypothesis and to evaluate the effect of biliary infection on common bile duct healing . A 3-mm longitudinal choledochotomy was performed in 120 rats and closed with a continuous 11/0 Ethilon suture . Common bile duct division with end-to-end anastomosis using interrupted 11/0 Ethilon sutures was performed in another 30 rats . Biliary infection was achieved in half of the animals with retrograde injection of living 046:K1:H31 strain Escherichia coli recovered from the rat colonic content . All rats with choledochotomy, including those with biliary infection, showed no bile leakage at the suture line, and the bursting pressure at the site of choledochotomy exceeded 40 mmHg as early as 24 h . Rats with common bile duct anastomosis alone showed no stricture formation for up to 6 months after operation . All rats with biliary sepsis developed complete occlusion at the anastomosis . On scanning electron microscopy, the biliary epithelium was well preserved in all rats . The study suggests that in rats with biliary sepsis the risk of bile leakage after primary closure of the common bile duct is negligible, but biliary infection may play a critical role in common bile duct stricture formation.

J Surg Res, 1991 Nov, 51(5), 413 - 6
Chylomicrons can inhibit endotoxin activity in vitro; Eichbaum EB et al.; Trauma, thermal injury, and nonlethal doses of endotoxin can promote the translocation of endotoxin across the mucosal barrier of the colon into the mesenteric lymphatics and systemic circulation . Bacterial endotoxemia induces changes in lipid metabolism, including an increase in circulating triglyceride-rich lipoproteins . Because cholesterol-rich lipoproteins can neutralize the toxic activity of endotoxin, both in vitro and in vivo, we asked whether triglyceride-rich chylomicrons can inhibit endotoxin activity in vitro as measured by a chromogenic Limulus assay . We tested the effect of intact versus heat-denatured chylomicrons on the in vitro activity of increasing concentrations of Escherichia coli (055:B5) endotoxin . Intact chylomicrons inhibited up to 12-fold the detection of as much as 1 microgram of endotoxin/mg of chylomicron triglyceride, compared to denatured chylomicrons (P less than 0.001) . This study shows that chylomicrons are potent inhibitors of endotoxin activity in vitro . Because translocated endotoxin from the colon associates with gut-derived chylomicrons in the mesenteric lymphatics, this may represent a natural defensive mechanism against endotoxemia of enteric origin.

DNA Cell Biol, 1991 Nov, 10(9), 695 - 8
Similarity between the amino-terminal portion of mammalian 58-kD sterol carrier protein (SCPx) and Escherichia coli acetyl-CoA acyltransferase: evidence for a gene fusion in SCPx; Baker ME et al.; A computer analysis of the amino acid sequences of rat and human 58-kD sterol carrier protein and Escherichia coli acetyl-CoA acyltransferase reveals that the two proteins have a segment of about 350 residues with strong sequence similarity . The ALIGN comparison scores for the rat and human sterol carrier proteins and the E . coli enzyme are 8.25 and 8.8 SD, respectively . The catalytically active cysteine of E . coli acetyl-CoA acyltransferase (cysteine 91) aligns with cysteine 93 and cysteine 94 on human and rat 58-kD sterol carrier protein, respectively.

Genetics, 1991 Nov, 129(3), 957 - 62
Transient mutators: a semiquantitative analysis of the influence of translation and transcription errors on mutation rates; Ninio J; A population of bacteria growing in a nonlimiting medium includes mutator bacteria and transient mutators defined as wild-type bacteria which, due to occasional transcription or translation errors, display a mutator phenotype . A semiquantitative theoretical analysis of the steady-state composition of an Escherichia coli population suggests that true strong genotypic mutators produce about 3 x 10(-3) of the single mutations arising in the population, while transient mutators produce at least 10% of the single mutations and more than 95% of the simultaneous double mutations . Numbers of mismatch repair proteins inherited by the offspring, proportions of lethal mutations and mortality rates are among the main parameters that influence the steady-state composition of the population . These results have implications for the experimental manipulation of mutation rates and the evolutionary fixation of frequent but nearly neutral mutations (e.g., synonymous codon substitutions).

Genetics, 1991 Nov, 129(3), 897 - 907
The selection-mutation-drift theory of synonymous codon usage; Bulmer M; It is argued that the bias in synonymous codon usage observed in unicellular organisms is due to a balance between the forces of selection and mutation in a finite population, with greater bias in highly expressed genes reflecting stronger selection for efficiency of translation . A population genetic model is developed taking into account population size and selective differences between synonymous codons . A biochemical model is then developed to predict the magnitude of selective differences between synonymous codons in unicellular organisms in which growth rate (or possibly growth yield) can be equated with fitness . Selection can arise from differences in either the speed or the accuracy of translation . A model for the effect of speed of translation on fitness is considered in detail, a similar model for accuracy more briefly . The model is successful in predicting a difference in the degree of bias at the beginning than in the rest of the gene under some circumstances, as observed in Escherichia coli, but grossly overestimates the amount of bias expected . Possible reasons for this discrepancy are discussed.

Anaesthesia, 1991 Nov, 46(11), 957 - 61
Environmental contamination during tracheal suction . A comparison of disposable conventional catheters with a multiple-use closed system device; Cobley M et al.; The extent of airborne environmental bacterial contamination which occurs following tracheal suction has been investigated in patients undergoing intermittent positive pressure ventilation in the intensive therapy unit . Two methods of performing suction, one using a conventional open technique and one using a closed system (Stericath), have been compared . Significantly lower levels of environmental contamination were observed when the closed system was used.

Acta Paediatr Scand, 1991 Nov, 80(11), 1019 - 24
Studies on adherence and outer membrane protein of enteropathogenic Escherichia coli 0127: H6 and their related plasmids; Wu SX et al.; The following characteristics were found in 20 epidemic strains of enteropathogenic Escherichia coli (EPEC) 0127: H6 isolated from an outbreak of neonatal diarrhea: 1) Absent Vero toxin production; 2) No potential for invasiveness; 3) Possession of 1.5 and 60 Md plasmid identical restriction digest and outer membrane protein (OMP) patterns; 4) Ability of localized adherence to HEp-2, HeLa and FL cells; 5) Capability to cause diarrhea in rabbits with destruction of the ileal microvilli at the areas of bacterial adherence . After elimination of the 60 Md plasmid from EPEC 0127: H6 the 45 and 82 Kd OMPs of the parent strain were lost . These plasmid-cured strains became non-adherent to HEp-2, HeLa and FL cells, and unable to cause diarrhea in rabbits . These results suggest that the pathogenic mechanism of EPEC 0127: H6 induced diarrhea may be related to the genes on a 60 Md plasmid expressed as 45 and 82 Kd OMPs which cause localized adherence to epithelial cells and destruction of ileal microvilli . This damage leads to a marked reduction in absorptive surface area, resulting in diarrhea.

Am Surg, 1991 Nov, 57(11), 701 - 5
Strategies in the management of pyogenic psoas abscesses; MacGillivray DC et al.; The presentation and management of eight patients with pyogenic psoas abscesses treated at the National Naval Medical Center, Bethesda, Maryland, between January 1986 and July 1989 are presented . The psoas abscesses were secondary to underlying gastrointestinal disease in six patients and sacral osteomyelitis in one patient . In one patient, the etiology of the abscess could not be determined . The average duration of symptoms in these patients was 16 days . Computed tomography was useful in identifying the abscess, defining its complexity, and planning therapy in all eight patients . Seven patients had complex, multiloculated abscesses, and one patient had a simple abscess . Extraperitoneal drainage was used in all patients . The patients with multiloculated abscesses had open surgical drainage, while the patient with the simple abscess had percutaneous catheter drainage . Most patients with a gastrointestinal etiology for their abscess underwent staged resection 3 to 6 weeks after the drainage procedure . There were no deaths, recurrent abscesses, or fistulae in these patients . Two patients developed thromboembolic complications postoperatively . Extraperitoneal drainage with staged resection of underlying gastrointestinal pathology is a safe and effective way of treating patients with psoas abscesses.

Mol Gen Genet, 1991 Nov, 230(1-2), 81 - 90
Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes; Gould JH et al.; Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants . Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA . The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin . Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA . This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein . Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity . This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified . We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli . Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.

Mol Gen Genet, 1991 Nov, 230(1-2), 75 - 80
Specificity of recA441-mediated (tif-1) mutational events; Yatagai F et al.; To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli . A 2.6-fold enhancement in lacI- mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced . G:C----C:G, G:C----T:A and A:T----T:A transversion events were specifically enhanced after SOS induction . A preferential 5'-Y-Purine-3' neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue) . In addition, a preference for transversion events at 5'-C/GTGG-3' sequences is also observed . Fifty events were recovered at the lacI "frameshift hotspot site" and were equally represented by 4 bp addition and deletion events . This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA+ background and is a consequence of the fourfold induction of the (-)4 event . This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI- mutations.

Mol Gen Genet, 1991 Nov, 230(1-2), 39 - 44
Cloning and expression analysis of a potato cDNA that encodes branching enzyme: evidence for co-expression of starch biosynthetic genes; Kossmann J et al.; One of the key enzymes involved in the formation of amylopectin, which is the major component of starch, is branching enzyme . A cDNA for potato branching enzyme was cloned by screening a tuber-specific cDNA expression library using an antiserum directed against a denatured preparation of the protein . Complementation of an Escherichia coli strain deficient in branching enzyme was achieved using a construct derived from this clone . Analysis of the expression of the gene in potato revealed a close association with conditions favouring starch biosynthesis . The expression pattern of the gene coding for potato branching enzyme, as analyzed at the mRNA level, closely resembles that of AGPase S, a gene coding for one of the subunits of ADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway . This raises the possibility that enzymes involved in the pathway are coordinately regulated at the transcriptional level.

Mol Gen Genet, 1991 Nov, 230(1-2), 332 - 6
Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences; Yamada H et al.; We previously demonstrated that the E . coli protein, H-NS (or H1a), encoded by the gene hns (or osmZ or bglY) preferentially recognizes curved DNA sequences in vitro . In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E . coli chromosome . The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns . It was further demonstrated that, in this hns deletion background, numerous E . coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.

Mol Gen Genet, 1991 Nov, 230(1-2), 28 - 32
Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli; Su TZ et al.; The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters . The regulation of ugp is mainly phoBR-dependent . Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression . Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation . Among different carbon sources tested, glucose caused the most complete repression . Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response . This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.

Mol Gen Genet, 1991 Nov, 230(1-2), 193 - 200
Concurrent transcription from the gid and mioC promoters activates replication of an Escherichia coli minichromosome; Ogawa T et al.; The origin of replication of the Escherichia coli chromosome (oriC) is located in an intercistronic region between the gidA and the mioC genes . The possibility that transcription from the promoters of these two genes is involved in minichromosome replication was examined . Inactivation of the gid promoter led to a reduction in transformation frequency with an oriC plasmid but inactivation of the mioC promoter did not . The decrease in transformation frequency was most pronounced when both promoters were inactive . Under conditions that selected for plasmid-harboring cells, mutation of the gid promoter caused efficient multimerization or integration of oriC plasmids into the chromosomal oriC region and loss of free plasmid molecules . These changes in plasmid structure were also observed, albeit less frequently, with some plasmids defective in mioC promoter activity . In an in vitro DNA replication system for oriC DNA, plasmids with a defective gid promoter had greatly reduced template activity and essentially no replication occurred when both promoters were inactive . These results suggest that coupled transcription starting from the gid as well as the mioC promoter activates initiation of plasmid replication, the major contribution being made by gid transcription . These two promoters are suggested to be under stringent control.

Mol Gen Genet, 1991 Nov, 230(1-2), 17 - 23
Growth phase-dependent modification of RNA polymerase in Escherichia coli; Ozaki M et al.; During the transition of Escherichia coli cultures from exponential growth to stationary phase, the pre-existing RNA polymerase was found to be converted into at least three different holoenzyme forms, which could be separated by phosphocellulose column chromatography . The relative levels of these three holoenzyme forms changed depending on the phase of cell growth . The altered stationary phase forms of RNA polymerase showed promoter recognition properties that were different from those of the holoenzyme from exponentially growing cells . Enzyme reconstitution experiments showed that the altered promoter selectivity was due to modification of the core enzyme . We propose that modulation of RNA polymerase plays a role in the global switch of gene expression during the transition from exponential growth to stationary phase.

J Endocrinol, 1991 Nov, 131(2), 219 - 27
Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins; Swennen D et al.; Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E . coli as inclusion bodies . These inclusion bodies were dissolved in 6 mol urea/l . Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules . Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography . The N-terminal sequence and bioactivities of both purified proteins were then analysed . Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium . When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites . The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

PCR Methods Appl, 1991 Nov, 1(2), 120 - 3
Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase; Nisson PE et al.; By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs . These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase . We have tested this method of ligation-independent cloning by using UDG to create complementary single-stranded sticky ends between vector and Alu-PCR products generated from cosmid clones containing DNA from human chromosome 21 . Using a single primer, Alu-PCR amplifies the sequence between appropriately oriented, repetitive (Alu) sequences in human DNA that are no more than 2 to 3 kb apart . Nineteen Alu-PCR products were observed in four human chromosome 21 cosmids . Thirteen of these products were detected among 48 subclones picked at random after cloning of the Alu-PCR products using UDG . The size or abundance of an Alu-PCR product did not appear to affect significantly the efficiency of cloning . Eight of the subclones were tested and all hybridized to human chromosome 21 DNA . UDG cloning should prove to be a general PCR cloning method that allows one to rapidly subclone small fragments from human genomic DNA.

Microb Pathog, 1991 Nov, 11(5), 379 - 85
Molecular cloning and expression of the 01 rfb region from a pyelonephritic Escherichia coli 01:H1:K7; Ding MJ et al.; The genes responsible for the biosynthesis of the O1 polysaccharide from a human pyelonephritic Escherichia coli were cloned and expressed in a rfb-deleted E . coli K-12 strain . Deletion analysis of the clone demonstrated that a DNA fragment size larger than 6.7 kb and smaller than 10 kb is responsible for O1-antigen biosynthesis.

Mol Biol (Mosk), 1991 Nov-Dec, 25(6), 1688 - 700
{Formation of phosphonoester bonds, catalyzed by DNA polymerases}; Diatkina NB et al.; 3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma-diphosphate (II) were synthesised . Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E . coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective . Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues . Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I . The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E . coli.

Mol Biol (Mosk), 1991 Nov-Dec, 25(6), 1615 - 25
{Properties of the replicator region of the natural plasmid pLG13 containing genes of the EcoRV restriction-modification system}; Zakharova MV et al.; The previously constructed plasmid pILRV8 that induces endonuclease EcoRV gene overexpression kills cells of some E . coli strains under the induction of this enzyme synthesis . Cell transformation by natural plasmid pLG13 carrying genes of the EcoRV restriction--modification system was found to appreciably enhance cell viability ("survival") under endonuclease overproduction . A plasmid pLG13 region located in immediate proximity to the methylase gene was shown to be responsible for the above effect . This region was also capable for autonomous replication . The analysis of the DNA primary structure in the found replicator region allowed to refer the pLG13 to ColE1 family plasmids . Perturbations in the region lead to loss of the "survival" effect and change of the plasmid replicative properties . A relationship between the replicon elements, the EcoRV genes region and "survival" effect is discussed . Based on the replicon found multicopy vector molecules have been constructed.

Mol Biol (Mosk), 1991 Nov-Dec, 25(6), 1580 - 7
{Hybridase cleavage of RNA . V . Complementary precision of cleavage}; Metelev VG et al.; The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E . coli RNase H was investigated . The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes . RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.

Thromb Haemost, 1991 Nov 1, 66(5), 569 - 74
Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator bm 06.022 in healthy volunteers; Martin U et al.; In a randomized, single-blind, placebo-controlled, cross-over Phase-I study pharmacokinetic and hemostatic properties of BM 06.022 were investigated in seven healthy, male human volunteers . The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 domain and the protease domain of human t-PA and is unglycosylated due to its expression in Escherichia coli cells . Vehicle or 6 MU (= 10.4 mg) BM 06.022 was administered as a single i.v . bolus injection of 10 ml over 2 min . BM 06.022 was well tolerated . Fibrinogen levels and clotting times remained unchanged at baseline levels after 6 MU BM 06.022; plasminogen and alpha 2-antiplasmin (collected on chloromethylketone) decreased maximally to 83 +/- 1% and 64 +/- 3%, respectively, of baseline . D-dimers and fibrinogen degradation products increased to 1,006 +/- 234 ng/ml and 555 +/- 155 ng/ml, respectively, after BM 06.022 . Half-life of BM 06.022-activity was 11.2 +/- 0.4 min and of antigen was 13.9 +/- 0.7 min, followed by a terminal half-life only for antigen of 173 +/- 33 min . Plasma clearance of BM 06.022 was 371 +/- 13 ml/min for activity and 183 +/- 15 ml/min for antigen . Thus, BM 06.022 is not fibrinogenolytic at 6 MU and is a fibrinolytic agent with a longer half-life than t-PA.

Br J Pharmacol, 1991 Nov, 104(3), 633 - 8
Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase; Mollace V et al.; 1 . Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1) . This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM) . 2 . The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E . coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h . This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS . Cycloheximide did not prevent the inhibitory activity of the non-treated cells . 3 . The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM) . Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity . This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM) . 4 . Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP) . L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cardiovasc Pharmacol, 1991 Nov, 18(5), 721 - 8
SK&F 86002 inhibits tumor necrosis factor formation and improves survival in endotoxemic rats; Smith EF 3rd et al.; We evaluated the effects of SK&F 86002, a dual 5-lipoxygenase/cyclooxygenase inhibitor, on the responses to 30 mg/kg Escherichia coli endotoxin (bacterial lipopolysaccharides, LPS) in conscious male Sprague-Dawley rats . Injection of LPS increased serum tumor necrosis factor (TNF alpha) activity from 20 U/ml (baseline) to 1,066 +/- 430 and 2,825 +/- 1,155 U/ml (n = 9) at 30 min and 1 h after administration of LPS, respectively (p less than 0.01 as compared with the vehicle control group) . This dose of LPS reduced the survival rate to 9% at 48 h (mean survival time 9.7 +/- 2.7 h), increased heart rate (HR) to 494 +/- 18 beats/min, increased the hematocrit to 56 +/- 2 vol%, reduced the circulating platelet count at 6 h to 40% of the initial value, and produced leukopenia of 30% of the initial value at 1 h, with recovery to the initial value at 6 h . Administration of 30 mg/kg SK&F 86002 (intragastric, i.g.) 1 h before injection of LPS reduced serum TNF alpha concentrations to 92 +/- 58 and 184 +/- 117 U/ml at 30 min and 1 h (p less than 0.05), respectively, and also reduced the hemoconcentration . The survival rate was improved to 60% (mean survival time 38.1 +/- 4.3 h; p less than 0.05), although there was no effect on the hemodynamic responses to LPS . SK&F 86002 significantly reduced thrombocytopenia at 30 min and 1 h (p less than 0.05), but not at 3 and 6 h, and had no effect on changes in white blood cell (WBC) count.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Res Hum Retroviruses, 1991 Nov, 7(11), 883 - 8
The DNA-dependent and RNA-dependent DNA polymerase activities of the reverse transcriptases of human immunodeficiency viruses types 1 and 2; Shaharabany M et al.; We have constructed a series of plasmids which, when introduced into Escherichia coli, induce the overexpression of soluble wild-type and mutated forms of the reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) . These proteins were analyzed previously for their RNA-dependent DNA polymerase (RDDP) and ribonuclease H (RNase H) activities . In the present study we assayed the different mutant RTs for their DNA-dependent DNA polymerase (DDDP) activity, employing an in situ polyacrylamide gel activity assay . The results indicate that both the RDDP and DDDP catalytic functions of HIV-1 RT mutants are affected similarly by mutations suggesting a high degree of overlap between the catalytic domains involved in both activities . Contrariwise, many of the HIV-2 RT mutants display no correlation between these two DNA polymerase activities, that is, the DDDP activity was not affected by the mutations introduced in the native enzyme in contrast to the RDDP activity . We were thus able to generate mutants of HIV-2 RT that unlike the wild-type RT, are capable of transcribing only DNA and not RNA . The disparity in mutational-catalytic relations between the two HIV-related RTs may reflect a possible difference in the structure and folding properties of the two proteins.

Mol Gen Genet, 1991 Nov, 230(1-2), 321 - 8
Regulation of replication of plasmid R1: an analysis of the intergenic region between copA and repA; Ohman M et al.; The synthesis of the rate-limiting RepA replication initiator protein of plasmid R1 is negatively controlled by an antisense RNA, CopA . The regulation is posttranscriptional and involves an inhibitory effect on RepA translation mediated by the binding of CopA to its target (CopT) in the leader region of the RepA mRNA . The evolutionary conservation of the intergenic region between the copA gene and the repA reading frame among plasmids related to R1 may be indicative of an important function in this regulation . One possibility is that sequences/structures in this region might be required for the presumed distal effect of CopAQCopT binding . We have performed a mutational analysis of this region, starting with a mutant repA-lacZ fusion plasmid that shows decreased RepA-LacZ synthesis compared to a wild-type construct, and have identified five compensatory mutations that increase repA-lacZ expression . Two of these were single base-pair substitutions in the copA promoter leading to a decrease in CopA transcription . The other three mutations increased RepA synthesis in the presence as well as in the absence of functional CopA . Reconstructed plasmids carrying these mutations--in conjunction with the original down-mutation or in an otherwise wild-type background--show the expected increase in copy number . The effect of two of these mutations is consistent with the destabilization of a putative secondary structure which may be responsible for the normally low translation rate of the RepA reading frame . The implications of the types of mutations found in this study, as well as the absence of other classes of mutations, are discussed in terms of alternative possible models of CopA-mediated inhibition of RepA synthesis.

Mol Gen Genet, 1991 Nov, 230(1-2), 230 - 40
Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli; Klein JR et al.; The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced . The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41,281 daltons) and EnvD (104,415 daltons) . The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane . The detection of a common transcript for both ORFs indicated the existence of an envCD operon . Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61 . Overproduction of EnvC protein appears to be lethal to Escherichia coli . The envD gene, however, could be cloned and expressed at high levels under control of the tac promoter without deleterious effects on the host.

J Cell Biol, 1991 Nov, 115(4), 1127 - 36
Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin; Murphy-Ullrich JE et al.; Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls . For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein . The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin . This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin . Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin . Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity . Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive . Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules . Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.

Mutat Res, 1991 Nov, 255(3), 281 - 91
Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells; Barrett SF et al.; Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive diseases with clinical and cellular hypersensitivity to UV radiation, differ in ability to repair UV DNA photoproducts in their overall genome: normal repair in CS, defective repair in XP . In order to characterize a DNA repair defect in an active gene in CS, we measured the capacity of cells from patients with CS and XP to reactivate 2 major types of UV-induced DNA damage, photoreactivatable (i.e., cyclobutane pyrimidine dimers) and non-photoreactivatable (primarily pyrimidine-(6-4)pyrimidone photoproducts), in the actively transcribing chloramphenicol acetyltransferase (cat) gene of the plasmid expression vector pRSV-cat . Epstein-Barr virus-transformed lymphoblast lines from 4 normal persons and from 3 patients with CS and from two with XP were transiently transfected with the plasmid, and the cat activity in cell extracts was determined . When the cells were transfected with UV-irradiated plasmid, expression was abnormally decreased in both the CS and XP cells . When the cyclobutane pyrimidine dimers in the UV-irradiated plasmid were removed by photoreactivation prior to transfection, cat expression in the CS, but not in the XP, lines reached normal levels . These data imply that both the XP and CS cells are unable to repair normally the cyclobutane pyrimidine dimer photoproducts which block transcription of cat . However, the CS, but not XP, cells can repair normally the other UV-induced photoproducts which block transcription . The ability of CS, but not XP, cells to repair these non-dimer photoproducts indicates that the active gene repair mechanism treats the cyclobutane pyrimidine dimer differently from the non-dimer photoproducts.

Mutat Res, 1991 Nov, 255(3), 265 - 71
Cross-adaptive response in Escherichia coli caused by pretreatment with H2O2 against formaldehyde and other aldehyde compounds; Nunoshiba T et al.; A cross-adaptive response (CAR), defined as a reduction of the effects of an agent by pretreatment with another agent, was demonstrated when E . coli WP2 cells were pretreated with hydrogen peroxide (H2O2) followed by challenging treatment with aldehyde compounds . Pretreatment with a sublethal dose (60 microM) of H2O2 for 30 min made WP2 cells resistant to the killing effects of formaldehyde (FA), and 4 other mutagenic aldehydes: glutaraldehyde, glyoxal, methyl glyoxal and chloroacetaldehyde . CAR was also observed in WP2uvrA (uvrA-) and ZA12 (umuC-) cells, but not in ZA60 (recA-) and CM561 (lexA- (Ind-} cells . A role of recA and lexA in CAR was further suggested by the lack of beta-galactosidase induction in recA- and lexA- cells by H2O2 . CAR and beta-galactosidase induction, however, were found to be separate events since CAR was recovered by introducing the recA+ gene into lexA- cells, but no induction of beta-galactosidase by H2O2 was observed in cells with the same gene transfer . These results suggest that H2O2 has the capacity to induce a function which reduces the killing effects of aldehydes, and the function is controlled by the recA gene without involvement of SOS response.

J Gen Virol, 1991 Nov, 72 ( Pt 11), 2747 - 55
Mapping of the epitopes of Epstein-Barr virus gp350 using monoclonal antibodies and recombinant proteins expressed in Escherichia coli defines three antigenic determinants; Zhang PF et al.; The Epstein-Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro . The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome . To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus . The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay . One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins . The data identify three antigenic determinants on gp350 . DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment . In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones . Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies.

J Bacteriol, 1991 Nov, 173(21), 6882 - 8
Procaine, a local anesthetic, signals through the EnvZ receptor to change the DNA binding affinity of the transcriptional activator protein OmpR; Rampersaud A et al.; Local anesthetics are known to reduce the level of OmpF and increase the synthesis of OmpC in the outer membrane of Escherichia coli K-12 . It has been shown that the anesthetics procaine and phenethyl alcohol (PEA) act at the transcriptional level for ompF and ompC and that in the case of procaine, its action is dependent on EnvZ, the membrane-bound signal transducer required for ompF and ompC expression . In an effort to further understand how anesthetics regulate ompF and ompC expression, we have analyzed the DNA binding properties of OmpR (the transcriptional activator protein for ompF and ompC genes) from cells treated with procaine or PEA . Treatment of a wild-type cell with either anesthetic converted OmpR from a low-affinity DNA binding form to a high-affinity DNA binding form . The change in DNA binding affinity was correlated with alterations in outer membrane porin profiles and could occur in the absence of protein synthesis . A strain lacking EnvZ was unable to respond to procaine to produce either the shift in the OmpR DNA binding property or cause any change in the outer membrane porin profile . PEA treatment was also dependent on EnvZ for the alteration in the OmpR DNA binding property, but it could induce ompC expression in the absence of EnvZ . Further studies suggest that the amino-terminal region of EnvZ is responsible for the procaine signalling . Our results indicate that procaine and PEA regulate ompF and ompC expression by modifying the DNA binding properties of OmpR through EnvZ signal transduction.

Eur J Biochem, 1991 Nov 1, 201(3), 653 - 9
Analysis of the complex transcription termination region of the Escherichia coli rrnB gene; Orosz A et al.; The complex terminator region of the Escherichia coli rrnB gene was analyzed by subcloning the terminators T1 and T2 and the inverted repeats IR1 and IR2 individually, or in various combinations, in a normal or inverted orientation into a terminator probe vector . The in vivo terminating efficiency was assayed by measuring the galactokinase activity encoded by the downstream galK gene . Termination efficiencies of all fragments were compared in two constructs, differing in the presence or absence of readthrough translation over the investigated terminator signal . The following main conclusions were drawn . (a) T1 and T2 are both efficient terminators in isolated forms . (b) IR1 and IR2 have some terminating effect (much lower than the proper terminators), especially in the inverted orientation . Their presence modifies the effect of the proper terminators in a quite unpredictable way, especially if these regions are translated . (c) The terminators are not symmetrical; in the inverted orientation T1 is practically inactive and T2 termination is reduced . (d) Translation radically decreases the efficiency of the terminators . (e) Several sequences in the rrnB gene, upstream of the terminator region (one in the 16S RNA and one in the 5S RNA coding region), are very efficient in vivo terminators in the inverted orientation.

J Virol, 1991 Nov, 65(11), 6024 - 30
Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody; Machida A et al.; The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene . By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no . 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised . The binding of no . 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated . Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope . No . 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them . Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c . Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.

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