Escherichia coli

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11466 - 70
Cloning of human acetyl-CoA carboxylase-beta and its unique features; Ha J et al.; Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids . In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles . In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis . ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the role of ACC-beta in controlling carnitine palmitoyltransferase I activity and fatty acid oxidation by mitochondria . If we exclude this unique sequence at the N terminus the two forms of ACC show about 75% amino acid identity . All of the known functional domains of ACC are found in the homologous regions . Human ACC-beta cDNA has an open reading frame of 7,343 bases, encoding a protein of 2,458 amino acids, with a calculated molecular mass of 276,638 Da . The mRNA size of human ACC-beta is approximately 10 kb and is primarily expressed in heart and skeletal muscle tissues, whereas ACC-alpha mRNA is detected in all tissues tested . A fragment of ACC-beta cDNA was expressed in Escherichia coli and antibodies against the peptide were generated to establish that the cDNA sequence that we cloned is that for ACC-beta.

Virology, 1996 Oct 15, 224(2), 548 - 54
Cloning and expression of a single-chain antibody fragment specific for foot-and-mouth disease virus; Mason P et al.; The gene for a single-chain antibody (VHK) to a conformational epitope on the type A12 foot-and-mouth disease virus (FMDV) particle was assembled and expressed in Escherichia coli . The VHK, purified from periplasmic extracts immunoprecipitated virus as efficiently as its parental monoclonal antibody (MAb) and exhibited the same binding specificity when tested against panel of natural and genetically engineered virus particles . The VHK neutralized type A12 virus in the presence of goat anti-mouse IgG; however, in the absence of the second antibody, only weak neutralizing activity was detected . Preliminary analysis of the mechanism of viral neutralization indicated that both the MAb and the VHK neutralize by the same mechanism . Small amounts of the VHK allowed infection of cells via Fc receptor-mediated adsorption in the presence of the second antibody . These data represent the first report of a single-chain neutralizing antibody for a picornavirus and provide insights into the mechanisms of viral neutralization and virus uptake.

Virology, 1996 Oct 15, 224(2), 368 - 79
Nla and Nlb of peanut stripe potyvirus are present in the nucleus of infected cells, but do not form inclusions; Hajimorad MR et al.; We investigated, by immunological and gene-fusion methods, whether the failure of peanut stripe potyvirus (PStV)-encoded nuclear inclusion proteins a (Nla) and b (Nlb) to form nuclear inclusions is due to the lack of their in vivo accumulation or the inability of one or both proteins to be transported into the nucleus Nla domains (Nla-VPg and Nla-proteinase), full-length Nlb, and full-length cylindrical inclusion (CI) protein of PStV were cloned, expressed in Escherichia coli, and used for antisera production . Immunoblot analysis of accumulation of Nla, Nlb, and CI in time course experiments revealed that they accumulated to similar levels in PStV-infected Nicotiana benthamiana . In immunocytochemical studies with electron microscopy, antiserum against Nla-VPg, Nla-Pro, and Nlb specifically labeled Nla and Nlb proteins throughout the nuclei of PStV-infected cells, in the absence of nuclear inclusions . Translational fusions were made between Nla and Nlb to either the green fluorescence protein or the beta-glucuronidase in vectors for transient gene expression or stable expression in transgenic plants respectively . Fusion proteins containing Nla accumulated in the nucleus, whereas fusion proteins containing Nlb accumulated in a punctate pattern in the cytoplasm . These data indicate that at least Nla possesses a nuclear localization signal.

Blood, 1996 Oct 15, 88(8), 3208 - 15
Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia; Manabe J et al.; Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II) . We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations . The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele . From this mutant allele, the product without the catalytic portion of the enzyme is generated . The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme . To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll . The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart . The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type . These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme . The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

Biochemistry, 1996 Oct 15, 35(41), 13485 - 93
A change in the internal aldimine lysine (K42) in O-acetylserine sulfhydrylase to alanine indicates its importance in transimination and as a general base catalyst; Rege VD et al.; O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes a beta-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide . The pyridoxal 5'-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine . In the present study, the Schiff base lysine was identified as lysine 42, and its role in the OASS reaction was determined by changing it to alanine using site-directed mutagenesis . K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates . Apo-K42A-OASS can be reconstituted with PLP, suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base . The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the alpha-aminoacrylate intermediate on addition of OAS, suggesting that K42 is involved in the abstraction of the alpha-proton in the beta-elimination reaction . The external aldimines formed upon addition of L-Ala or L-Ser are stable and represent a tautomer that absorbs maximally at 420 nm, while L-Cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, and is also seen in the overall reaction after addition of primary amines to the assay system . The use of a small primary amine such as ethylamine or bromoethylamine in the assay system leads to the initial formation of an internal (gamma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of OAS . Activity could not be fully recovered, and only a single turnover is observed . Data suggest a significant rate enhancement resulting from the presence of K42 for transimination and general base catalysis.

Biochemistry, 1996 Oct 15, 35(41), 13363 - 7
Chemical rescue of Asp237-->Ala and Lys358-->Ala mutants in the lactose permease of Escherichia coli; Frillingos S et al.; Asp237 (helix VII) and Lys358 (helix XI) form a salt bridge in the lactose permease, and neutral replacement of either residue inactivates . Remarkably, noncovalent neutralization of the unpaired Asp or Lys residue, respectively, with n-alkylsulfonates or n-alkylamines of appropriate size restores active transport to high levels in the mutants . Saturation with respect to the concentration of the alkylamines and different size preferences suggest that the alkylamines bind sterically at position 358 . Rescue of Asp237-->Ala by alkylsulfonates is apparently more indiscriminate, since methane-, ethane-, or propane-sulfonate have comparable effects . Sodium and chloride, respectively, are also effective in rescuing the Lys358-->Ala and Asp237-->Ala mutants, while various other compounds are ineffective . In marked contrast to Asp237-->Ala or Lys358-->Ala permease, alkylsulfonates or alkylamines have no effect whatsoever on the activity of mutants with neutral replacements for Asp240, Glu269, Arg302, Lys319, His322, or Glu325 . The results support the conclusion that neutral replacement of one member of the charge pair between Asp237 and Lys358 leads to inactivation because of an unpaired charge in the low dielectric of the membrane . In addition, the findings are consistent with the idea that interactions between Arg302 and Glu325, His 322 and Glu269, and Asp240 and Lys319 play important roles in the mechanism of the permease, which is not the case for either Asp237 or Lys358 or the salt bridge between the two residues.

Biochemistry, 1996 Oct 15, 35(41), 13303 - 9
DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata; Pommier Y et al.; Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata . It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent . The present study investigates the interactions of ecteinascidin 743 with DNA . Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications . Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA . However, DNA denaturation reversed the DNA modifications . The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine . Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2 . Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA) . The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter . A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere . Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity . Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine . These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.

Biochemistry, 1996 Oct 15, 35(41), 13294 - 302
Interaction of pyridine nucleotide substrates with Escherichia coli dihydrodipicolinate reductase: thermodynamic and structural analysis of binary complexes; Reddy SG et al.; E . coli dihydrodipicolinate reductase exhibits unusual nucleotide specificity, with NADH being kinetically twice as effective as NADPH as a reductant as evidenced by their relative V/K values . To investigate the nature of the interactions which determine this specificity, we performed isothermal titration calorimetry to determine the thermodynamic parameters of binding and determined the three-dimensional structures of the corresponding enzyme-nucleotide complexes . The thermodynamic binding parameters for NADPH and NADH were determined to be Kd = 2.12 microM, delta G degree = -7.81 kcal mol-1, delta H degree = -10.98 kcal mol-1, and delta S degree = -10.5 cal mol-1 deg-1 and Kd = 0.46 microM, delta G degree = -8.74 kcal mol-1, delta H degree = -8.93 kcal mol-1, and delta S degree = 0.65 cal mol-1 deg-1, respectively . The structures of DHPR complexed with these nucleotides have been determined at 2.2 A resolution . The 2'-phosphate of NADPH interacts electrostatically with Arg39, while in the NADH complex this interaction is replaced by hydrogen bonds between the 2' and 3' adenosyl ribose hydroxyls and Glu38 . Similar studies were also performed with other pyridine nucleotide substrate analogs to determine the contributions of individual groups on the nucleotide to the binding affinity and enthalpic and entropic components of the free energy of binding, delta G degree . Analogs lacking the 2'-phosphate containing homologs . For all analogs, the total binding free energy can be shown to include compensating enthalpic and entropic contributions to the association constants . The entropy contribution appears to play a more important role in the binding of the nonphosphorylated analogs than in the binding of the phosphorylated analogs.

Biochemistry, 1996 Oct 15, 35(41), 13282 - 7
A zinc binding site in viral serine proteinases; De Francesco R et al.; The NS3 protein of hepatitis C virus contains a chymotrypsin-like serine proteinase domain . We built a homology model of this domain which predicts the presence of a tetradentate metal binding site formed by three cysteines and one histidine . These residues are strictly conserved in all known hepatitis C viral genotypes as well as in other recently discovered related hepatitis viruses . We show that the hepatitis C virus enzyme does indeed contain a Zn2+ ion with S3N ligation and that the metal is required for structural integrity and activity of the enzyme . Strikingly, the residues forming the metal binding site are also conserved in the chymotrypsin-like 2A cysteine proteinases of picornaviruses . Remarkably, in these highly variable viral genomes the metal binding site is more conserved than the catalytic residues and thus allows us to define a novel class of zinc binding chymotrypsin-like proteinases and to identify a new attractive target for antiviral therapy.

Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 334 - 9
Mutagenic specificity of N-methyl-N'-nitro-N-nitrosoguanidine in the tonB gene on the chromosome of Escherichia coli recA+ and recA- cells; Wang X et al.; DNA base sequence changes induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis has been determined for the endogenous tonB gene of Escherichia coli recA+ strain and its isogenic recA56 strain . In the recA+ strain, base substitutions accounted for 48 mutations among 54 MNNG-induced independent mutations analyzed and consisted of 45 G:C to A:T transitions, two A:T to G:C transitions and one A:T to C:G transversion . In the recA56 strain, 67% (34/51) were base substitutions among which G:C to A:T transition (28/51) predominated, followed by three A:T to T:A transversions, two G:C to T:A transversions, and one A:T to G:C transition . These mutagenic specificities were consistent with the mispairing predicted by the methylation of the O6 position of guanine . In both strains the G:C to A:T mutations were found at guanine residues preceded by either guanine or thymine on the non-transcribed strand of the target gene.

Biochim Biophys Acta, 1996 Oct 11, 1313(3), 179 - 86
Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties; Rhyner JA et al.; Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified . Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet . of 4.9 x 10(3) M-1 . The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet . of 3.4 to 4.6 x 10(3) M-1, i.e . very similar to those of PVwt . The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively . The K'Mg.compet values of these mutants are about 4-times lower than in PVwt . The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+ . After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm . Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+ . Specific antibodies against human alpha-parvalbumin were raised in rabbits . Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.

J Mol Biol, 1996 Oct 11, 262(5), 746 - 55
Glycinamide ribonucleotide transformylase undergoes pH-dependent dimerization; Mullen CA et al.; Glycinamide ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5) . We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state . In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering . The data give a best fit to a cooperative three-proton transfer mechanism: {formula: see text} A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization . Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization . Fitting of the normalized titration curves yields an apparent pKa = 7.16(+/-0.02) and a subnanomolar KD for the transition . Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization . There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization . Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution . Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell.

J Mol Biol, 1996 Oct 11, 262(5), 706 - 20
The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2; Kauppi B et al.; The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 A resolution using molecular replacement and refined to an R-value of 19.1% (Rfree = 25%) with good stereo-chemistry . The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coli R2 . However, several important structural differences are observed . Unlike the E . coli protein, the mouse dimer is completely devoid of beta-strands . The sequences differ significantly between the mouse and E . coli R2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence . Therefore, the three-dimensional structures of other mammalian ribonucleotide reductase R2 proteins are expected to be very similar to that of the mouse enzyme . In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein . In E . coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine . These structural variations may explain the observed differences in sensitivity to radical scavengers . The structure determination is based on diffraction data from crystals grown at pH 4.7 . Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites . This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position . Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.

J Mol Biol, 1996 Oct 11, 262(5), 615 - 28
Identification of the bases in the ompF regulatory region, which interact with the transcription factor OmpR; Huang KJ et al.; Expression of the outer membrane protein OmpF of Escherichia coli K-12 is influenced by a variety of environmental signals . Most of the signals are thought to regulate OmpF expression at the level of transcription initiation . A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF . In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompF regulatory region that are in contact with OmpR . Our results indicate that the -107 to -39 region of ompF contains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules . We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes . Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides . Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site . Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites . Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompF and serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals.

J Med Chem, 1996 Oct 11, 39(21), 4162 - 6
Inactivation of S-adenosyl-L-homocysteine hydrolase by amide and ester derivatives of adenosine-5'-carboxylic acid; Wnuk SF et al.; S-Adenosyl-L-homocysteine (AdoHcy) hydrolase has been shown to have (5'/6') hydrolytic activity with vinyl (5') or homovinyl (6') halides derived from adenosine (Ado) . This hydrolytic activity is independent of its 3'-oxidative activity . The vinyl (or homovinyl) halides are converted into 5'(or 6')-carboxaldehydes by the hydrolytic activity of the enzyme, and inactivation occurs via the oxidative activity . Amide and ester derivatives of Ado-5'-carboxylic acid were prepared to further probe the hydrolytic capability of AdoHcy hydrolase . The oxidative activity (but not the hydrolytic activity) is involved in the mechanism of inhibition of the enzyme by the ester and amide derivatives of Ado-5'-carboxylic acid, in contrast to the inactivation of this enzyme by adenosine-derived vinyl or homovinyl halide analogues during which both activities are manifested.

J Biol Chem, 1996 Oct 11, 271(41), 25624 - 9
Different inducibility of expression of the two xylanase genes xyn1 and xyn2 in Trichoderma reesei; Zeilinger S et al.; Regulation of formation of the extracellular xylanase system of Trichoderma reesei QM 9414 during growth on xylan, cellulose, and replacement onto a number of soluble inducers was investigated by Northern analysis of xyn1 and xyn2 transcripts and by the use of the Escherichia coli hph (hygromycin B-phosphotransferase-encoding) gene as a reporter . Whereas the xyn1 promoter is active in the presence of xylan and xylose, and virtually silenced in the presence of glucose, the xyn2 promoter enables basal transcription at a low level, but is enhanced in the presence of xylan and xylobiose and also of sophorose or cellobiose . The respective regulatory nucleotide regions were localized on a 221-base pair fragment and a 55-base pair fragment of the xyn1 and xyn2 5'-upstream noncoding sequences, respectively . Electrophoretic mobility shift assays, using cell-free extracts, identified induction-specific protein-DNA complexes: one complex of high mobility was observed under basal, noninduced conditions (glucose) with xyn2, which was in part replaced by a slow-migrating complex upon induction by xylan or sophorose . Both complexes bound to a CCAAT box . With xyn1, the induced complex also binds to a CCAAT box, but this binding is not observed in the presence of the carbon catabolite repressor Cre1, which binds to a nearby located consensus motif.

J Biol Chem, 1996 Oct 11, 271(41), 25575 - 81
Stonustoxin is a novel lethal factor from stonefish (Synanceja horrida) venom . cDNA cloning and characterization; Ghadessy FJ et al.; Stonustoxin (SNTX) is a multifunctional lethal protein isolated from venom elaborated by the stonefish, Synanceja horrida . It comprises two subunits, termed alpha and beta, which have respective molecular masses of 71 and 79 kDa . SNTX elicits an array of biological responses both in vitro and in vivo, particularly a potent hypotension that appears to be mediated by the nitric oxide pathway . As a prelude to structure-function studies, we have isolated and sequenced cDNA clones encoding the alpha- and beta-subunits of SNTX from a venom gland cDNA library . The deduced amino acid sequence of neither subunit shows significant homology with any known protein . Protein sequence alignment does, however, show the subunits to be 50% homologous to each other and implies that they may have arisen from a common ancestor . The subunits of this novel toxin lack typical N-terminal signal sequences commonly found in proteins that are secreted via the endoplasmic reticulum-Golgi apparatus pathway, indicating the possibility of its being secreted by a non-classical pathway, which is not clearly understood . The SNTX subunits have been expressed in Escherichia coli as cleavable fusion proteins that cross-react with antibodies raised against the native toxin . To the best of our knowledge, this is the first complete sequence of a fish-derived protein toxin to be reported.

J Biol Chem, 1996 Oct 11, 271(41), 25423 - 9
Identification of the cpdA gene encoding cyclic 3',5'-adenosine monophosphate phosphodiesterase in Escherichia coli; Imamura R et al.; We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC) . The expression of beta-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene . Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells . Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene . We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP . In addition, we showed that the CpdA protein hydrolyzed cAMP to 5'-adenosine monophosphate and that its activity was activated by iron . Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase.

J Biol Chem, 1996 Oct 11, 271(41), 25360 - 8
A partially functional DNA helicase II mutant defective in forming stable binary complexes with ATP and DNA . A role for helicase motif III; Brosh RM Jr et al.; To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine . UvrDD248N failed to form stable binary complexes with either DNA or ATP . However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present . The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP . The mutant protein also demonstrated a reduced DNA unwinding activity . The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase . The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase . The uvrDD248N allele was also characterized in genetic assays . The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II . In contrast, the mutant protein only partially complemented the mutator phenotype . A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed.

J Biol Chem, 1996 Oct 11, 271(41), 25247 - 52
Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump; Li J et al.; The ArsA ATPase is the catalytic subunit of the Ars pump that catalyzes arsenical extrusion in Escherichia coli, thus providing resistance . The active form of ArsA is a homodimer with four nucleotide binding sites, two from each monomer . The codons for Gly-15 in the N-terminal consensus nucleotide binding sequence and Gly-334 in the C-terminal sequence were individually mutated to cysteine codons . Cells expressing an arsAG334C mutation retained arsenite resistance, while an arsAG15C mutation resulted in substantial reductions in arsenite resistance, transport, and ATPase activity . Selection for suppression of the G15C mutation that restored arsenite resistance yielded an A344V substitution . Ala-344 is located adjacent to the C-terminal nucleotide binding sequence . The second site mutation did not suppress the loss of resistance resulting from G18D, G20S, or T22I substitutions in the N-terminal nucleotide binding site . Cells expressing the G15C/A344V double mutant regained arsenite extrusion . These results suggest a spatial proximity of Gly-15 and Ala-344 and support a model for interaction of the nucleotide binding sites in ArsA.

J Biol Chem, 1996 Oct 11, 271(41), 25178 - 83
Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein; Mizushima T et al.; DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP . We earlier reported that 3-acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K . (1994) Bioorg . Med . Chem . Lett . 4, 1771-1774) . In the present study, derivatives of 3-acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined . Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations . Among the derivatives, 3-{N-(11-carboxyundecyl)}carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 microM . The mode of the inhibition was competitive . We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins . This inhibition was specific for the initiation of DNA replication rather than for the elongation . The inhibition was overcome by preincubation of DnaA protein with ATP . Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system . We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.

J Biol Chem, 1996 Oct 11, 271(41), 25126 - 30
Identification of histone H2A.X as a growth factor secreted by an androgen-independent subline of mouse mammary carcinoma cells; Watabe Y et al.; Shionogi carcinoma 115 (SC 115) cells and Chiba subline 2 (CS 2) cells are clones of an androgen-responsive mouse tumor cell line and its autonomous subline, respectively . We have shown previously that CS 2 cells produce a heparin-binding growth factor that stimulates the growth of SC 115 cells as well as the growth of themselves . In this study, a growth factor was purified from serum-free conditioned media of CS 2 cells cultured without testosterone . A heparin-binding fraction showed growth- promoting activity on SC 115 cells and BALB/3T3 cells . The amino acid sequence analysis revealed that the components were identical to histones H2A.1 and H2A.X . Since histone H2A purified from bovine thymus had almost no growth-promoting activity on SC115 cells, histone H2A.X was assumed to be a growth factor . cDNA of histone H2A.X was cloned from a library of CS 2 cells, and its sequence was confirmed . The expressed product of histone H2A.X cDNA in Escherichia coli showed remarkable stimulatory effects on growth of SC 115 cells cultured in the absence of testosterone . These results indicate that histone H2A.X is secreted from CS 2 cells cultured without testosterone and plays a role as a growth factor.

J Biol Chem, 1996 Oct 11, 271(41), 25083 - 8
Heterologous expression of three plant serpins with distinct inhibitory specificities; Dahl SW et al.; For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed . BSZx, BSZ4, and WSZ1 were assayed for inhibitory activity against trypsin, chymotrypsin, and cathepsin G, and cleavage sites in the reactive center loop were identified by sequencing . BSZx proved to be a potent inhibitor with specific, overlapping reactive centers either at P1 Arg for trypsin or at P2 Leu for chymotrypsin . At 22 ;C, the apparent rate constant for chymotrypsin inhibition at P2 (ka = 9.4 x 10(5) M-1 s-1) was only four times lower than for trypsin at P1 (ka = 3.9 x 10(6) M-1 s-1), and the apparent inhibition stoichiometries were close to 1 . Furthermore, our data suggest that cathepsin G was inhibited by BSZx (ka = 3.9 x 10(6) M-1 s-1) at both the P1 Arg and P2 Leu . These results indicate a unique adaptability of the reactive center loop of BSZx . WSZ1 inhibited chymotrypsin (ka = 1.1 x 10(5) M-1 s-1) and cathepsin G (ka = 7.6 x 10(3) M-1 s-1) at P1 Gln and not, as for BSZx, at the more favorable P2 Leu . BSZ4 inhibited cathepsin G (ka = 2.7 x 10(4) M-1 s-1) at P1 Met but was hydrolyzed by trypsin and chymotrypsin . The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data.

J Biol Chem, 1996 Oct 11, 271(41), 25079 - 82
Selectivity of the renal collecting duct water channel aquaporin-3; Echevarria M et al.; Aquaporin-3 (AQP3) is a water channel found in the basolateral cell membrane of principal cells of the renal collecting tubule as well as in other epithelia . To examine the selectivity of AQP3, the permeability to water (Pf), urea (Pur), and glycerol (Pgly) of Xenopus oocytes injected with cRNA encoding AQP3 was measured . Oocytes injected with cRNA encoding either human or rat aquaporin-1 (AQP1) were used as controls . Although both aquaporins permit water flow across the cell membrane, only AQP3 was permeable to glycerol and urea (Pgly > Pur) . The uptake of glycerol into oocytes expressing AQP3 was linear up to 165 mM . For AQP3 the Arrhenius energy of activation for Pf was 3 kcal/mol, whereas for Pgly and Pur it was >12 kcal/mol . The sulfhydryl reagent p-chloromercuriphenylsulfonate (1 mM) abolished Pf of AQP3, whereas it did not affect Pgly . In addition, phloretin (0.1 mM) inhibited Pf of AQP3 by 35%, whereas it did not alter Pgly or Pur . We conclude that water does not share the same pathway with glycerol or urea in AQP3 and that this aquaporin, therefore, forms a water-selective channel.

J Biol Chem, 1996 Oct 11, 271(41), 25063 - 6
Nucleotides reveal polynucleotide phosphorylase activity from conventionally purified GroEL; Ybarra J et al.; GroEL, as conventionally purified, can be incubated with nucleotides to produce high molecular weight material with an absorption maximum at 260 nm . This material is most clearly demonstrated when samples are subjected to gel filtration under conditions where GroEL is monomeric . There is a time-dependent increase in the high molecular weight material that occurs on incubation with ADP or, more slowly, with ATP . This material is generated during incubation, and none is present in the initial samples . Experiments with nucleases, proteases, radiolabeled nucleotides, and chemical cleavage reagents demonstrate that the high molecular weight material is polyadenylic acid whose formation is inhibited by phosphate . These results are consistent with the GroEL samples containing polynucleotide phosphorylase activity . Nondenaturing gels stained with acridine orange, after incubation in ADP, reveal that the activity producing the poly(A) coelectrophoreses with authentic polynucleotide phosphorylase . Conditions that remove the tryptophan-like fluorescence from preparations of GroEL also remove the PNPase activity . Thus, this activity is not associated with GroEL itself . The results are consistent with reports that GroEL can associate with RNase E and with other studies showing that RNase E and PNPase can form complexes . Thus, the present experiments support suggestions that GroEL can participate in multiprotein complexes that are involved in mRNA processing and degradation.

Gene, 1996 Oct 10, 175(1-2), 223 - 31
Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans; Trueman LJ et al.; The crnA nitrate transporter from Aspergillus nidulans was identified as belonging to the major facilitator superfamily (MFS) of membrane transporters . Degenerate oligonucleotides corresponding to the crnA sequences at the locations of two conserved sequence motifs were designed and used in the polymerase chain reaction (PCR) to amplify related sequences from barley root poly(A)+ RNA . A 130 bp cDNA fragment with sequence similarities to crnA was amplified and used as a probe to screen a barley root cDNA library . Two full-length clones (pBCH1 and pBCH2) were isolated . The nt sequences of pBHC1 and pBCH2 are closely related (80% identical) and potentially encode hydrophobic polypeptides of 54.7 and 55.0 kDa respectively, with twelve predicted transmembrane domains . The encoded polypeptides are 41-43% identical to the A . nidulans CRNA protein and 56-57% identical to NAR-3, a high-affinity nitrate transporter from the eukaryotic alga Chlamydomonas reinhardtii . Phylogenetic analysis indicated that crnA, nar-3 and the barley homologues belong to a new family within the MFS, a family that also includes narK, the gene for a nitrite efflux pump in Escherichia coli . In northern blots, BCH1 hybridised to a mRNA species of 1.9 kb which is rapidly induced in barley roots by NO3-, but not by NH4+, and genomic Southern blots indicated that there may be seven to ten BCH1-related genes in the barley genome.

Gene, 1996 Oct 10, 175(1-2), 199 - 201
The 5' regulatory region from the Drosophila pseudoobscura hsp82 gene results in a high level of reporter gene expression in Lucilia cuprina embryos; Coates CJ et al.; We have previously examined the efficiency of two Drosophila melanogaster promoters to enable reporter gene expression in embryos of the Australian sheep blowfly, Lucilia cuprina . Both the hsp70 heat-shock promoter and the actin5C promoter resulted in low levels of expression of a reporter gene in these embryos . In this study, the D . pseudoobscura hsp82 promoter (phsp82) was tested for its ability to direct the expression of the Escherichia coli chloramphenicol acetyltransferase-encoding gene (cat) . We report that the level of CAT activity in L . cuprina embryos was comparable to that obtained with the same construct in D . melanogaster, indicating that phsp82 functions efficiently in this non-drosophilid insect . The results suggest that phsp82 may be utilised in other non-drosophilid insects in which poor expression levels are obtained from constructs containing the hsp70 or actin5C promoters.

Gene, 1996 Oct 10, 175(1-2), 83 - 7
Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification; Fujisaki S et al.; A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli . The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303 . The gene mapped at 10.9 min on the E . coli chromosome and was designated fsr (fosmidomycin resistance) . Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa . A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol . Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments . The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr . The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid . These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E . coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Nature, 1996 Oct 10, 383(6600), 550 - 3
Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans; Goedert M et al.; The paired helical filament (PHF) is the major component of the neurofibrillary deposits that form a defining neuropathological characteristic of Alzheimer's disease . PHFs are composed of microtubule-associated protein tau, in a hyperphosphorylated state . Hyperphosphorylation of tau results in its inability to bind to microtubules and is believed to precede PHF assembly . However, it is unclear whether hyperphosphorylation of tau is either necessary or sufficient for PHF formation . Here we show that non-phosphorylated recombinant tau isoforms with three microtubule-binding repeats form paired helical-like filaments under physiological conditions in vitro, when incubated with sulphated glycosaminoglycans such as heparin or heparan sulphate . Furthermore, heparin prevents tau from binding to microtubules and promotes microtubule disassembly . Finally, we show that heparan sulphate and hyperphosphorylated tau coexist in nerve cells of the Alzheimer's disease brain at the earliest known stages of neurofibrillary pathology . These findings, with previous studies which show that heparin stimulates tau phosphorylation by a number of protein kinases, indicate that sulphated glycosaminoglycans may be a key factor in the formation of the neurofibrillary lesions of Alzheimer's disease.

Biochemistry, 1996 Oct 8, 35(40), 13222 - 30
Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain; Huang JF et al.; Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD) . The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor . To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no . L14771) was made as a fusion protein in Escherichia coli . We show here that a truncated CDPK lacking a CaM-LD (e.g . mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM) . We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction . When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses . When the junction and CaM-LD are tethered in a single polypeptide (e.g . in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g . the tethered CaM-LD cannot bind a separate junction) . A mutation which disrupts the putative CaM-LD binding sequence (e.g . substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding . This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation . Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism . CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

Biochemistry, 1996 Oct 8, 35(40), 13180 - 5
Different sensitivities to acid denaturation within a family of proteins: implications for acid unfolding and membrane translocation; Evans LJ et al.; Colicins A, B, and N form a family of membrane pore-forming toxins with > 50% sequence identity in their toxic C-terminal domains . The colicin A C-terminal domain has been shown to insert into model membranes via an acidic molten-globule insertion intermediate, and thus this family provides a means to compare acid unfolding of related proteins . Unlike the domains of colicins A and B which are acidic, that of colicin N is very basic with fewer Asp and Glu residues . If surface positive charge density is the crucial factor in acidic molten globule formation, colicin N should begin to unfold at higher pH values than colicins A or B . However, comparison of their CD spectra reveals that colicins A and B both form acidic molten globules but colicin N does not . None of the proteins forms a denaturant-induced molten globule at neutral pH where the proteins exhibit very similar stabilities . The acidic unfolding cannot therefore be due to excess positive surface charge and may be caused by a subset of acidic residues as has been predicted for myoglobin . The difference between the colicins is confirmed by their in vivo membrane insertion, with colicins A and B inserting much faster than colicin N . Stopped-flow circular dichroism measurements of colicin A insertion into vesicles confirmed that a molten globule insertion intermediate occurs at the membrane surface.

Biochemistry, 1996 Oct 8, 35(40), 13147 - 56
Substrate specificity of Escherichia coli MutY protein; Bulychev NV et al.; The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA . In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme . In contrast to a recent report {Lu, A.-L., et al . (1995) J . Biol . Chem . 270, 23582}, dA:8-oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (kcat/Km) for dA:8-oxo-dG and dA:dG of 39 600 x 10(-6) and 383 x 10(-6) (min-1 nM-1), respectively . kcat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0 . The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested . Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 x 10(-6) (min-1 nM-1), whereas replacement of dA by 2'-O-methyladenosine virtually abolished enzymatic activity . Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2'-O-methylguanosine generated a substrate with a low specificity constant . Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate . Duplexes containing a carbocyclic analog of dA were not cleaved . A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme.

Biochemistry, 1996 Oct 8, 35(40), 12993 - 3000
Folding intermediates of a beta-barrel membrane protein . Kinetic evidence for a multi-step membrane insertion mechanism; Kleinschmidt JH et al.; The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood . A key question is whether folding and insertion are coupled or separable processes . We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy . OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles . In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range . The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature . The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C) . The activation energy for this process was 46 +/- 4 kJ/mol . A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures . These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers . We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures . These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers {Rodionova et al . (1995) Biochemistry 34, 1921-1929} . Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA . On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.

FEBS Lett, 1996 Oct 7, 394(3), 316 - 20
Quaternary structure of human nucleoside diphosphate kinase isoforms HA and HB in solution; Schaertl S; Human isoforms of nucleoside diphosphate kinase, NDPK-HA and NDPK-HB, have been expressed in E . coli and purified . Their apparent molecular masses have been determined by FPLC gel filtration . Absolute molecular masses were measured by equilibrium ultracentrifugation and sedimentation coefficients determined from the sedimentation velocity . Under near-physiological conditions, NDPK-HA has a mass of 101 +/- 3 kDa, close to that calculated for a hexamer (102.11 kDa), whilst NDPK-HB has a mass of 71 +/- 3 kDa, close to a tetramer (68.67 kDa) . The sedimentation coefficients, 5.15 +/- 0.2 and 3.41 +/- 0.1 x 10(-13) s, for HA and HB also indicate a hexamer and a tetramer respectively . This suggests, although the crystal structure shows a hexameric quaternary arrangement {Webb et al . (1995) J . Mol . Biol . 251, 574-587}, that NDPK-HB forms tetramers in solution like bacterial NDPK {Williams et al . (1993) J . Mol . Biol . 234, 1230-1247}.

FEBS Lett, 1996 Oct 7, 394(3), 311 - 5
Loop mutations affect ferritin solubility causing non-native aggregation of subunits or precipitation of fully assembled polymers; Jappelli R et al.; As a consequence of elevated expression rates, the intracellular aggregation of polypeptide chains is commonly observed in E . coli . Although wild-type human ferritin, a polymeric iron storage protein, accumulates in the soluble form at high level in the bacterial cytoplasmic fraction, some amino acid substitutions in an exposed loop direct the synthesis of a highly insoluble product . We found that two mechanisms can lead to the aggregation of ferritin . While some mutations prevent ferritin polymerisation, others cause the precipitation of molecules in the assembled state.

J Mol Biol, 1996 Oct 4, 262(4), 437 - 58
Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA; Auxilien S et al.; In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg . The corresponding tRNA genes all have adenosine at position 34 . Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34 . The homologous E . coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG . The base conversion occurs by a hydrolytic deamination-type reaction . This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in {18O}water . This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast . It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA . The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture . Enzymatic recognition of tRNA does not depend on the presence of any "identify" nucleoside other than adenosine 34 . Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis . However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35 . Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase . Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34 . An apparent K(m) of 2.3 nM for its natural substrate tRNASer (anticodon AGA) was measured . Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast.

J Mol Biol, 1996 Oct 4, 262(4), 407 - 12
High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli; Calderone TL et al.; The expression of eukaryotic genes in Escherichia coli is one of the most frequently used tools of modern science . The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E . coli . We report here 36 to 42% misincorporation of lysine at three AGA codons in a well-expressed protein . This misincorporation yields a protein whose electrospray mass spectrum (ESMS) shows peaks at the expected mass (M), M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7), 37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity, respectively . Replacement of either all three AGA codons or the two closest to the 3' end of the gene by the more common CGC arginine codon gave a protein with a single ESMS peak . Misincorporation could also be eliminated by the co-expression of the tRNA(UCL)Arg gene, argU . These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought.

J Mol Biol, 1996 Oct 4, 262(4), 389 - 95
The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17; Bomer U et al.; The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date . We report that the essential subunit Tim17 is highly conserved in evolution . The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria . Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface . In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23 . The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation.

J Chromatogr A, 1996 Oct 4, 746(1), 17 - 24
Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption; Seeger A et al.; A two-step chromatographic procedure for purification of basic fibroblast growth factor (bFGF) from high-cell-density cultures of recombinant E . coli is described . Heparin-Sepharose as a material which shows a high affinity to endothelial growth factors was used as sorbent for purification of bFGF from the soluble cell fraction . A one-step affinity chromatographic procedure resulted in very pure bFGF . However, this one-step affinity isolation of bFGF caused the loss of around 60% of the recombinant protein . A combination of ion-exchange chromatography with heparin-Sepharose affinity chromatography was favored for bFGF purification . A first cation-exchange chromatographic step resulted in a solution of bFGF with a purity of around 70% . The weak cation exchanger CM Sepharose C50 was preferred in comparison to the strong cation exchanger S-Sepharose because of the higher recovery of bFGF . With the ion-exchange chromatographic step prior to the heparin-Sepharose affinity chromatography, the total yield of recovery of bFGF increased to 56% compared to 40% using the one-step purification procedure with heparin-Sepharose . To characterize the equilibrium parameters of adsorption, batch experiments for the calculation of maximum capacities and dissociation constants for CM-Sepharose C50 and heparin-Sepharose were carried out . The equilibrium experiments revealed that adsorption of bFGF to the ion-exchange sorbent followed single-site interaction according to the Langmuir model of adsorption . The adsorption of bFGF to heparin-Sepharose was described by a double Langmuir approach of two independent binding sites with different maximum capacities and dissociation constants . The purified bFGF showed a high biological activity and circular dichroic spectra of a proper folded molecule . The analysis of the N-terminal amino acid sequence revealed a mixture of two fractions of bFGF, which both are characterized by the cleavage of the first amino acid methionine . In addition, half of the bFGF molecules lacked the second amino acid alanine.

Cell, 1996 Oct 4, 87(1), 115 - 25
A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy; Jain C et al.; A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein . By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev . All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev . The spatial constraints suggested by these data have enabled us to model the structure of this complex.

J Biol Chem, 1996 Oct 4, 271(40), 25027 - 34
Characterization of truncated forms of the KdpD protein, the sensor kinase of the K+-translocating Kdp system of Escherichia coli; Puppe W et al.; The expression of the kdpFABC operon, coding for the K+-translocating Kdp system, is controlled by the two regulatory proteins, KdpD and KdpE, which belong to the group of sensor kinase/response regulator systems . This study describes the construction and analysis of KdpD sensor kinases, in which different deletions in the N-terminal part of the protein were introduced . Truncated KdpD proteins, in which the membrane-spanning segments were deleted, had lost their phosphorylation capacity . Truncated KdpD proteins, in which the four membrane-spanning helices were untouched, were still phosphorylated, and the phosphoryl group could be transferred to the response regulator KdpE in vitro . Furthermore, these truncated KdpD proteins cause dephosphorylation of KdpE(P), which is comparable with that of the wild-type protein . To investigate the effect of the deletions on signal transduction in vivo the corresponding kdp genes were transferred to the chromosome . Growth studies with the mutant strains are in accord with the data obtained from the in vitro studies . Furthermore, kdp expression was investigated using a KdpA-LacZ fusion . The data obtained support the notion that the extent of kdp expression is modulated by the N-terminal part of KdpD.

J Biol Chem, 1996 Oct 4, 271(40), 24954 - 61
A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity; Minnick DT et al.; In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V . and Steitz, T.A . (1993) Science 260, 352-355) . The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb . The mutant polymerase has relatively normal dNTP binding and catalytic rate . However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced . Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs . The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage . These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA . The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.

J Biol Chem, 1996 Oct 4, 271(40), 24862 - 8
DNA binding by cut homeodomain proteins is down-modulated by protein kinase C; Coqueret O et al.; The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats . Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains . Using nuclear extracts from mammalian cells, Cut-specific DNA binding was increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo . Sequence analysis of Cut repeats revealed the presence of sequences that match the consensus phosphorylation site for protein kinase C (PKC) . Therefore, we investigated whether PKC can modulate the activity of mammalian Cut proteins . In vitro, a purified preparation of PKC efficiently phosphorylated Cut repeats, which inhibited DNA binding . In vivo, a brief treatment of cells with calphostin C, a specific inhibitor of PKC, led to an increase in Cut-specific DNA binding, whereas phorbol 12-myristate 13-acetate, a specific activator of PKC, caused a decrease in DNA binding . The PKC phosphorylation sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Thr415, Thr804, and Ser987 within Cut repeats 1-3, respectively . Cut homeodomain proteins were previously shown to function as transcriptional repressors . Activation of PKC by phorbol 12-myristate 13-acetate reduced transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites was not affected . Altogether, our results indicate that the transcriptional activity of Cut proteins is modulated by PKC.

J Biol Chem, 1996 Oct 4, 271(40), 24806 - 10
Heat shock-induced DNA relaxation in vitro by DNA gyrase of Escherichia coli in the presence of ATP; Kataoka K et al.; Genetic studies revealed that DNA gyrase seems to catalyze immediate and transient DNA relaxation after Escherichia coli cells are exposed to heat shock (Ogata, Y., Mizushima, T., Kataoka, K., Miki, T., and Sekimizu, K . (1994) Mol . Gen . Genet . 244, 451-455) . We have now obtained biochemical evidence to support this hypothesis . DNA gyrase catalyzed an increase in the linking number of DNA and relaxation of negatively supercoiled DNA, under physiological concentrations of ATP . Analyses by gel filtration chromatography of each subunit revealed that DNA relaxation activity co-migrated with each subunit . The linking number of DNA increased as the temperature increased . Further, the reaction was inhibited by nalidixic acid or by oxolinic acid . Based on these results, we propose that DNA gyrase participates in a concerted reaction with DNA topoisomerases in the immediate relaxation of DNA in cells exposed to heat shock.

J Biol Chem, 1996 Oct 4, 271(40), 24736 - 40
Identification of residues of spinach thioredoxin f that influence interactions with target enzymes; Geck MK et al.; The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood . To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes . Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77 . Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared . All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax . In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins . The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f . Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity.

J Biol Chem, 1996 Oct 4, 271(40), 24720 - 7
Localization of the effector-specifying regions of Gi2alpha and Gqalpha; Medina R et al.; Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes . Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins . To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits . The chimeras were generated using an in vivo recombination method in Escherichia coli . The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain . An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase . This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP . Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C . Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain.

J Biol Chem, 1996 Oct 4, 271(40), 24662 - 9
Mechanism of translesion DNA synthesis by DNA polymerase II . Comparison to DNA polymerases I and III core; Paz-Elizur T et al.; Bypass synthesis by DNA polymerase II was studied using a synthetic 40-nucleotide-long gapped duplex DNA containing a site-specific abasic site analog, as a model system for mutagenesis associated with DNA lesions . Bypass synthesis involved a rapid polymerization step terminating opposite the nucleotide preceding the lesion, followed by a slow bypass step . Bypass was found to be dependent on polymerase and dNTP concentrations, on the DNA sequence context, and on the size of the gap . A side-by-side comparison of DNA polymerases I, II, and III core revealed the following . 1) Each of the three DNA polymerases bypassed the abasic site analog unassisted by other proteins . 2) In the presence of physiological-like salt conditions, only DNA polymerase II bypassed the lesion . 3) Bypass by each of the three DNA polymerases increased dramatically in the absence of proofreading . These results support a model (Tomer, G., Cohen-Fix, O . , O'Donnell, M., Goodman, M . and Livneh, Z . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 1376-1380) by which the RecA, UmuD, and UmuC proteins are accessory factors rather than being absolutely required for the core mutagenic bypass reaction in induced mutagenesis in Escherichia coli.

J Biol Chem, 1996 Oct 4, 271(40), 24649 - 54
Escherichia coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT family of proteins . Cloning, purification, and characterization of the enzyme; O'Handley SF et al.; The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the beta-phosphorus to produce dAMP and inorganic pyrophosphate . It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg2+, a Km of 0.8 mM and a kcat of 5.2 s-1 at 37 degrees C for dATP . dAMP is a weak competitive inhibitor with a Ki of approximately 4 mM, while PPi is a much stronger inhibitor with an apparent Ki of approximately 20 microM . The enzyme contains the highly conserved signature sequence GXVEX2ETX6REVXEEX2I designating the MutT family of proteins . However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT- mutator phenotype, and thus must serve a different biological role in the cell.

J Biol Chem, 1996 Oct 4, 271(40), 24604 - 9
Modeling of a mutation responsible for human 3-hydroxy-3-methylglutaryl-CoA lyase deficiency implicates histidine 233 as an active site residue; Roberts JR et al.; 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine . Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target . Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines . These observations prompted a functional test of the importance of His-233 . The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized . In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets . Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude . Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity . This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites . In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.

J Biol Chem, 1996 Oct 4, 271(40), 24449 - 57
A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain; Preugschat F et al.; The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity . The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion . The enzyme was not selective for the NTP substrate . For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme . The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA) . Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence . The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 microM at pH 7.0 and 25 degrees C . The enzyme had maximal affinity for NA with 12 or more nucleotides . A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector . The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s-1 and 2.7 s-1, respectively . The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 microM-1 s-1 (3.9 s-1) and 290 microM-1 s-1 (2.27 s-1), respectively . The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 microM-1 s-1 (<0.5 s-1) and 0.9 microM-1 s-1 (<10(-1) s-1), respectively . These data were consistent with a random kinetic mechanism.

J Biol Chem, 1996 Oct 4, 271(40), 24349 - 52
Cloning and functional analysis of the beta-carotene hydroxylase of Arabidopsis thaliana; Sun Z et al.; An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxylase was identified by functional complementation in Escherichia coli . The product of this cDNA adds hydroxyl groups to both beta rings of the symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin (beta,beta-carotene-3,3'-diol) and converts the monocyclic beta-zeacarotene (7',8'-dihydro-beta,psi-carotene) to hydroxy-beta-zeacarotene (7',8'-dihydro-beta,psi-carotene-3-ol) . The epsilon rings of delta-carotene (epsilon,psi-carotene) and alpha-zeacarotene (7',8'-dihydro-epsilon,psi-carotene) are poor substrates for the enzyme . The predicted amino acid sequence of the A . thaliana enzyme resembles the four known bacterial beta-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids . Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E . coli . Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate beta-cryptoxanthin (beta,beta-carotene-3-ol), predominantly, when beta-carotene was provided as the substrate . It is suggested that amino acid residues 70-129 of the A . thaliana enzyme may play a role in formation of a functional homodimer.

Gene, 1996 Oct 3, 174(2), 293 - 7
Sequence analysis of the 2,4-dichlorophenol hydroxylase gene tfdB and 3,5-dichlorocatechol 1,2-dioxygenase gene tfdC of 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011; Koiv V et al.; Chlorocatechol 1,2-dioxygenase (CC12O) and 1,2-dichlorophenol hydroxylase (DCPH) encoding genes tfdC and tfdB are located on a 4.2-kb DNA fragment cloned from the 2,4-dichlorophenoxyacetic acid (2,4D) degrading plasmid pEST4011 . The nucleotide sequences of tfdC and tfdB were determined . The DCPH is coded by a 1758-bp gene and CC12O is coded by a 762-bp gene . The deduced M(r) of these proteins are 64.09 kDa and 28.2 kDa, respectively . Expression analysis of tfdB and tfdC in Escherichia coli suggested that these genes form one operon, tfdCB.

Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 211 - 5
Electron paramagnetic resonance (EPR) studies on hydrogenase-1 (HYD1) purified from a mutant strain (AP6) of Escherichia coli enhanced in HYD1; DerVartanian ME et al.; Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2 . Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1 . EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species . Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals . At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared . The detection of a 4Fe-4S cluster signal was noted . Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear . The 4Fe-4S complex intensity was slightly increased . The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.

Parasite Immunol, 1996 Oct, 18(10), 507 - 13
Host-protective fragments and antibody binding epitopes of the Taenia ovis 45W recombinant antigen; Lightowlers MW et al.; The protective efficacy of a recombinant Taenia ovis vaccine antigen, 45W, was compared in sheep vaccine trials with antigen expressed by the full length 45W cDNA and by incomplete copies of the cDNA . Vaccine, trials were also carried out using antigen expressed by a cDNA (45S) having a sequence similar, but not identical, to 45W . Stability of the 45W antigen expressed in Escherichia coli was found to be increased after deletion of cDNA sequence encoding 19 COOH-terminal amino acids . This truncated form of the antigen was designated 45WB/X . Vaccination of sheep with antigen expressed by 45W, 45WB/X, as well as full length 45W and 45S cDNAs, induced high levels of protection . Vaccination with antigen expressed by an incomplete copy of the 45S cDNA clone did not induce protection . Comparison of deduced amino acid sequences for these clones suggests that the host-protective epitope(s) of the 45W antigen occur on either or both of the 23 and 9 amino acid peptides at the amino and carboxyl termini of 45W, respectively . Antibody binding epitopes of 45W were investigated in ELISA using overlapping 9 amino acid peptides . Protection was found to correlate with the induction of antibody to two 9 amino acid peptides.

Braz J Med Biol Res, 1996 Oct, 29(10), 1317 - 20
Distinct antigenic subtypes of human beta interferon can be distinguished by neutralization; Golgher RR et al.; Different molecular configurations of human beta interferon were titrated with the standard reference antiserum of the National Institutes of Health (NIH) which had been prepared with natural beta fibroblast interferon in order to determine to what extent differences in these configurations would influence the neutralization of the antiviral action of interferon . Neutralization tests were carried out in Vero cells by diluting both interferon and antiserum . Encephalomyocarditis virus was employed as challenge virus . The neutralization titer was considered to have been reached when the effect of eight units of interferon was reduced to one . Two natural beta interferons prepared from fibroblasts and from amniotic membranes gave similar high titers . However, titers were reduced five-fold with recombinant interferons expressed in Escherichia coli, which do not contain carbohydrate, one with the natural sequence and a mutant with a single amino acid substitution (cysteine for serine) . The NIH antiserum did not neutralize the effect of a protein fraction from amniotic membranes antigenically different from the human alpha, beta or gamma interferons but having the biological activity of interferon . We conclude that the carbohydrate moieties of human beta interferons are essential for their recognition by the NIH antiserum and that antibodies specific for human recombinant beta interferon, which does not contain carbohydrate, are needed.

Res Microbiol, 1996 Oct, 147(8), 609 - 13
The qmeA (ts) mutation of Escherichia coli is localized in the fabI gene, which encodes enoyl-ACP reductase; Kleerebezem M et al.; The phenotypes of temperature-sensitive qmeA and fabI mutants of Escherichia coli appear to be very similar . Furthermore, the qmeA mutation could be complemented by the fabI gene on a plasmid, and the fabI allele derived from the qmeA mutant strain harbours a nucleotide substitution identical to that from a previously characterized fabI mutant . These results show that the qmeA gene is, in fact, identical to the fabI gene, which encodes enoyl-ACP reductase, involved in fatty acid elongation.

Res Microbiol, 1996 Oct, 147(8), 597 - 608
In vivo positive effects of exogenous pyrophosphate on Escherichia coli cell growth and stationary phase survival; Biville F et al.; We have studied the effect of exogenous pyrophosphate on growing cells of Escherichia coli . In the presence of 10 mM of pyrophosphate, the entry into the stationary phase was delayed and thus a significant increase in the growth yield was observed (25 to 35%) when the bacteria were grown in glucose minimal medium . Furthermore, the synthesis of 52 polypeptides was affected, as demonstrated by two-dimensional electrophoresis . Among the 22 proteins identified by comparison with the E . coli gene-protein index and/or by microsequencing procedures, 15 were involved either in catabolic or anabolic pathways of the intermediary metabolism or in stress responses . Subsequent physiological experiments enabled us to conclude that pyrophosphate exerted a direct or indirect effect on bacterial growth by (1) conferring upon cells a better capacity to use carbon sources and (2) inducing biosynthetic processes . Finally, we show that exogeneous pyrophosphate enhanced the stationary phase survival of E . coli cells.

Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 109 - 22
The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli; Leont'eva O et al.; The kinetics of Photinus pyralis and Luciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible lambda PR promoter in Escherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay . E . coli cells were cultivated at temperature schemes 28-42-21 degrees C or 28-21 degrees C, or at alkali pH shift . In the cases of thermoinduction and pH shift, the luciferase expressions have similar features . The 3-h thermoinduction (42 degrees C) followed by the incubation at 21 degrees C, for 10 h resulted in the maximal amount of the luciferase protein of 4-5% of the total cell proteins . The yield did not change further . The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21 degrees C and reached the maximum after 50-60 h when the synthesized luciferase protein adopted the native-like conformation . At the same time, only 50% of the latter appeared to be catalytically active . An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content . Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.

Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 97 - 107
HIV-I protease . Cloning, expression, and purification; Dergousova NI et al.; A new method for obtaining HIV-I protease was suggested . Fusion proteins composed of the N-terminal fragment of human gamma-interferon and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed in Escherichia coli cells . The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein . Protein I was cleaved by enterokinase . The solubility of protein I was increased by treating with Na-sulfite/Na-tetrathionate under denaturing conditions . Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found . The hydrolysis products were separated by reversed-phase FPLC . The amount of tryptophan and cysteine residues in the enzyme obtained was estimated . The activity of HIV-I protease was determined using the chromogenic peptide . AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of beta-galactosidase and a fragment of gag proteins, including p17-p24 processing site.

Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 85 - 96
Protein engineering of de novo protein with predesigned structure and activity; Dolgikh DA et al.; The de novo protein albebetin has been engineered (J . Mol . Biol . 1992, 225, 927-931) to form a predesigned tertiary fold that has not yet been observed in natural proteins . Analysis of albebetin expressed in a cell-free system and in Escherichia coli revealed its compactness, relative stability, and the secondary structure close to the predesigned one . The blast-transforming biological activity of human interferon was grafted to albebetin by attachment of an eight amino acid interferon fragment to the N-terminus of albebetin next to its first methionine residue . The chimeric protein was expressed in a wheat germ cell-free translation system and tested for its structural properties, receptor binding, and biological activity . According to the tests, albebetin incorporating the active interferon fragment has a compact and relatively stable structure, and binds the murine thymocyte receptor effectively . It activates the blast transformation reaction of thymocyte cells even more efficiently than human interferon at low concentrations.

Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 47 - 56
D-glyceraldehyde-3-phosphate dehydrogenase . Properties of the enzyme modified at arginine residues; Nagradova NK et al.; Examination of the properties of Escherichia coli and rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction . The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case of E . coli enzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH . These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries . Modification of the arginine procedures marked changes in the shape of the charge transfer complex spectrum in the region of 300-370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD(+), although the coenzyme binding characteristics remain largely unaltered . On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction.

Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 13 - 23
Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli . Substrate specificity and stability under irradiation; Mareeva EA et al.; Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41-->His and Phe143-->Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation . Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism . Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring . The replacement of Phe41 with the ionizable His residue destabilizes the enzyme . The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals . The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and O-phenylene diamine . The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring . The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine.

Genetika, 1996 Oct, 32(10), 1326 - 32
{Identification and expression of plasmid genes participating in the synthesis of microcin C51}; Fomenko DE et al.; A structural gene of heptapeptide, which is a component of the microcin C51 molecule, was identified by hybridization of plasmid DNA fragments and a mixture of synthesized oligonucleotides with the sequence corresponding to that of amino acids in the peptide . Sequence analysis of the structural gene of microcin peptide and its promoter region was performed . The data obtained indicate that the peptide of microcin C51 is synthesized on ribosomes . Four polypeptides of 67, 39, 16, and 14 kDa were identified using the system of minicells . These polypeptides are specified by a DNA fragment responsible for microcin synthesis and immunity in a producer cell . Apparently, three of these polypeptides with molecular masses of 67, 39, and 16 kDa are responsible for microcin production and immunity . The 67 kDa polypeptide is involved in the expression of immunity to microcin and, probably, in microcin production.

Mol Immunol, 1996 Oct, 33(15), 1171 - 6
Ezrin, radixin and moesin are possible auto-immune antigens in rheumatoid arthritis; Wagatsuma M et al.; In order to deduce which cellular molecules react with the sera from patients with rheumatoid arthritis (RA), human and mouse cellular extracts were fractionated stepwise, by ethanol precipitation and their reactivity analysed by Western blotting . It was found that three cytoplasmic molecules with molecular weights of 80,000, 81,000 and 77,000 were immunoreactive and they were identified as ezrin (E), radixin (R), and moesin (M), respectively, by partial amino acid sequencing . Using cDNA clones of these human molecules, recombinant proteins were produced in Escherichia coli and used to enable the antigens to detect the antibodies in the sera of patients with RA . Of 71 sera tested, 24 sera (33.8%) reacted with at least one of three recombinant antigens, although there was no significant correlation between the presence of the antibodies and clinical manifestations, such as disease duration or stage . There was also no discernible relationship to other auto-antibodies such as antinuclear antibodies (ANA) and rheumatoid factor . The results suggest that ERM proteins are possible novel auto-immune target antigens for RA.

Proc Natl Sci Counc Repub China B, 1996 Oct, 20(4), 110 - 6
Expression of polyphosphate kinase inhibits the glucose uptake in Escherichia coli; Liu JK et al.; This paper examines the effects of phosphate pool and expression of polyphosphate kinase on glucose uptake by expressing the polyphosphate kinase under the control of lac promoter . The E . coli transformant of pL1, containing an IPTG controllable element for polyphosphate kinase expression, showed that the total intracellular phosphate significantly increased . However, the rate of glucose uptake by the resting plasmid-bearing cells with IPTG induction significantly decreased . These findings suggest that the polyphosphate can not directly function as an energy source in E . coli or at least not as a good energy supplier.

Mol Immunol, 1996 Oct, 33(14), 1119 - 25
The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein; Marrugo J et al.; The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5 . Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1 . Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs . When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis . The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis . It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes . CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

Mol Immunol, 1996 Oct, 33(14), 1113 - 8
Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen; Chatel JM et al.; The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients . Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously {Cho et al . (1994) J . Biol . Chem . 269, 11 102-11 107} . The method has been modified by adding an immunoaffinity purification step . A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced . rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge . In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95% . All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding . No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG . In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG . In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients . The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Can J Physiol Pharmacol, 1996 Oct, 74(10), 1141 - 8
Muscle aspartyl protease (cathepsin D) activity: detection using a chromophoric substrate and relation to wasting in DBA/2 mice implanted with leukemic L1210 tumor cells; Bolger GT et al.; The relationship between skeletal muscle aspartyl protease activity (APA) and wasting was investigated in male DBA/2 mice inoculated with L1210 tumor cells . Using the peptidic substrate H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH, which is specific for aspartyl proteases, proteases, proteolytic activity was detected in a number of tissues including muscle by using a crude extraction procedure for isolation of lysosomal enzymes . Biochemical characterization and increased muscle levels following either fasting or injection of endotoxin (ETX) suggest that the enzyme is likely cathepsin D . The wasting syndrome accompanying the tumor was measured by comparing the weight of the skinned hind limb in treated and control animals . DBA/2 mice inoculated intraperitoneally with L1210 cells developed multiple solid tumors in the peritoneum and ascites; maximal tumor burden was reached by 16 days . There was a significant reduction in hind limb weight (16 +/- 2%; mean +/- SE) and significant increase (31 +/- 8%) in muscle APA associated with the development of ascites and solid tumors . Plasma APA activity was substantially increased (240 +/- 33%), while liver and spleen APA were increased (10-20%) but not significantly . Chronic pepstatin administration, 30 mg.kg-1.day-1, for 7 days concurrent with the initiation of observable ascites and solid tumor formation (7 days post-inoculation), completely inhibited hind limb weight loss and alleviated the tumor-dependent increase of APA in both plasma and muscle without altering tumor development . Delaying the administration of pepstatin by 3 days resulted in less of an inhibition (33 +/- 13%) of hind limb weight loss . Thus, cathepsin D or a similar aspartyl protease appears to be of key importance in the wasting syndrome associated with cachexia.

Food Chem Toxicol, 1996 Oct, 34(10), 959 - 62
Lethality induced by stannous chloride on Escherichia coli AB1157: participation of reactive oxygen species; Dantas FJ et al.; Stannous chloride (SnCl2) has been widely used in nuclear medicine as a reducing agent of pharmaceutical products radiolabelled with technetium-99m . To verify whether the lethality induced by this salt could be mediated by reactive oxygen species (ROS), Escherichia coli cultures were treated with SnCl2 in the presence of catalase, ROS scavengers or metal-ion chelators . The inactivation effect, as measured by survival determination, was abolished by thiourea, sodium benzoate, dipyridyl or catalase . The results suggest the participation of ROS, generated by a Fenton-like reaction, in the lethal effect induced by SnCl2.

Biokhimiia, 1996 Oct, 61(10), 1758 - 71
{Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography}; Guzov VM et al.; Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity . To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified . Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography . Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane . Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system . Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography . Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone . Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system . Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively . According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.

An Esp Pediatr, 1996 Oct, 45(4), 398 - 402
{Risk and outcome factors in necrotizing enterocolitis}; Carbonell Estrany X et al.; OBJECTIVE: The objective of this study was to identify risk and outcome factors in necrotizing enterocolitis (NEC) . PATIENTS AND METHODS: We have studied 72 cases of NEC collected from 1987 until 1994 in the three hospitals of the integrated Unit . A case-control study matched for gestational age and center was performed for 26 risk factors . Conditional logistic regression was used in significant bivariate variables . The 18 outcome factors had the same statical treatment, but without the paired design . RESULTS: Serous infections previous to NEC, apnea and feeding increments greater than 20 cc/kg/day have been identified as risk factors for preterm babies (p < 0.05) . Severe acidosis and pneumoperitoneum have been found significant outcome variables, but with very low discriminatory capacity . CONCLUSIONS: It has been found difficult to identify risk factors for NEC besides the gestational age . Outcome factors have very low sensitivity . Preventive treatment should be directed to decrease the effect of the inflammatory mediators in the gastrointestinal tract.

Berl Munch Tierarztl Wochenschr, 1996 Oct, 109(10), 385 - 7
{Practical experiences in the therapy of postweaning edema disease in piglets}; Papp I et al.; In a field trial 23 weeks old weaned piglets were orally inoculated with E . coli O139 K12 H1 . The piglets received the same food and were kept under the same management regime . They were selected randomly into four groups and treated after the first clinical signs of CNS involvement of edema disease as follows during three days: Group 1: received a single daily i.m . application of 4 mg/kg body weight Melperone . Group 2: received a single daily i.m . application of 2 mg/kg body weight Amperozide . Group 3: was treated orally (intranasally) with 2 mg/kg body weight Amphetamin in a single daily application . Group 4: untreated control . The following parameters were evaluated before the begin of the therapy (day 0-4) . A: Occurrence of diarrhea in a group B: Food consumption per piglet per day C: Death of piglets per group After the begin of the therapy (day 5-32) D: Death of piglets per group E: Average daily feed intake per piglet The results showed that the Melperone and Amphetamin treatment was superior regarding all examined parameters when compared to Amperozide treatment and to the control group.

Comput Appl Biosci, 1996 Oct, 12(5), 415 - 22
Syntactic recognition of regulatory regions in Escherichia coli; Rosenblueth DA et al.; MOTIVATION: One of the most common methodologies to identify cis-regulatory sites in regulatory regions in the DNA is that of weight matrices, as testified by several articles in this issue . An alternative to strengthen the computational predictions in regulatory regions is to develop methods that incorporate more biological properties present in such DNA regions . The grammatical implementation presented in this paper provides a concrete example in this direction . RESULTS: On the basis of the analysis of an exhaustive collection of regulatory regions in Escherichia coli, a grammatical model for the regulatory regions of sigma 70 promoters has been developed . The terminal symbols of the grammar represent individual sites for the binding of activator and repressor proteins, and include the precise position of sites in relation to transcription initiation . Combining these symbols, the grammar generates a large number of different sentences, each of which can be searched for matching against a collection of regulatory regions by means of weight matrices specific for each set of sites for individual proteins . On the basis of this grammatical model, a Prolog syntactic recognizer is presented here . Specific subgrammars for ArgR, LexA and TyrR were implemented . When parsing a collection of 128 sigma 70 promoter regions, the syntactic recognizer produces a much lower number of false-positive sites than the standard search using weight matrices.

J Photochem Photobiol B, 1996 Oct, 36(1), 47 - 53
A mutant of Escherichia coli hyper-resistant to a number of DNA damaging agents: location of the mutational site; Ahmad SI; A mutant of Escherichia coli, isolated as hyper-resistant to UVC, is found to be hyper-resistant to UVA, H2O2, low concentrations of nalidixic acid, novobiocin, UVA plus H2O2 and UVA plus 8-methoxypsoralen . A mutational site (uvh) conferring the hyper-resistance phenotype to UVC, and presumably to other DNA damaging agents, has been mapped at the 89.9 min region on the chromosome . Complementation analysis with an F-prime uvh+/uvh- diploid strain showed that the uvh+ allele is dominant over uvh- in trans . Studies with a variety of plasmids, carrying various LexA regions, introduced into the UV hyper-resistant strain show that mutation at the uvh locus may be responsible for derepression of the SOS inducible repair system . Based on the results, it is suggested that uvh is a part of the SOS inducible system . A plausible explanation for the hyper-resistance phenotypes for various DNA damaging agents and a model for the genetic control of a second set of putative SOS regulons are presented.

Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1681 - 5
A novel gene that interferes with the phosphotransfer signal transduction mediated by the EnvZ osmosensor in Escherichia coli; Hirokawa K et al.; In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli . A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes . In this study, an E . coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E . coli cells by a multicopy plasmid . The gene product was identified as F107, which was previously characterized as a hypothetical protein in E . coli genome databases . F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III . The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants . These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase . Possible mechanisms by which F107 affects the EnvZ function are discussed.

Int J Androl, 1996 Oct, 19(5), 271 - 7
Influence of Escherichia coli on motility parameters of human spermatozoa in vitro; Diemer T et al.; The influence of E . coli on human sperm motility was studied in vitro . Semen samples were prepared by a swim-up technique and adjusted to 22 x 10(6) spermatozoa/ml . Samples were then inoculated with different concentrations of a uropathogenic strain of E . coli, serotype 06, with initial sperm/bacteria ratios varying between 10:1 and 10000:1 . Motion parameters were analysed by computer-aided motility analysis directly, and 2, 4 and 6 h after inoculation . In a second series of experiments, bacterial replication was inhibited by addition of chloramphenicol . In a third series, the effect of E . coli culture filtrates on sperm motility was investigated . The direct inhibitory effect of E . coli on progressive motility of spermatozoa was found to depend upon the bacterial concentration . A distinct inhibitory effect was observed only at a sperm/bacteria ratio of approximately 1, achieved by growth of E . coli during the experiments . For modality of motion, no distinct changes were observed . When growth of bacteria was prevented by chloramphenicol, no inhibitory effect on sperm motility was detected . Sperm motility was not inhibited by E . coli culture filtrates . Analysis by electron microscopy revealed multiple adhesions of E . coli to spermatozoa, causing variable ultrastructural damage as probable morphological correlates of immobilization.

Eur J Haematol, 1996 Oct, 57(4), 278 - 85
Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system; Asami N et al.; The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector . Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86 . It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR . In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ . For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture . The BM cells were selected with G418, and transferred into irradiated recipient mice . LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.

Avian Dis, 1996 Oct-Dec, 40(4), 927 - 30
Large plasmids of avian Escherichia coli isolates; Doetkott DM et al.; The plasmid DNA of 30 Escherichia coli isolates from chickens was extracted and examined using techniques designed to isolate large plasmids . This plasmid DNA was examined for the presence of certain known virulence-related genes including cvaC, traT, and some aerobactin-related sequences . Seventeen of the 30 isolates contained from one to four plasmids greater than 50 kb in size . Eleven of these 17 strains possessed plasmids greater than 100 kb in size . Therefore, E . coli isolates of chickens frequently contain large plasmids, and many of these plasmids are likely to contain virulence-related sequences.

Avian Dis, 1996 Oct-Dec, 40(4), 865 - 74
Ornithobacterium rhinotracheale infection in turkey breeders; De Rosa M et al.; A sequence of outbreaks of respiratory disease separated by intervals of about 2 wk occurred on three turkey breeder ranches . The last two ranches affected belonged to different companies and were separated by a distance of 11 km . Mortality on the last ranch was particularly severe among certain segregated groups of turkeys that included toms, heavier birds, and birds undergoing a stressful event such as artificial insemination . On this ranch, percentages of mortality within an 18-day period were 5.2% in toms, 2.4% in hens, 7.4% in heavy toms and 5.4% in heavy hens . Turkeys from 27 to 42 wk of age were examined during the outbreaks . Gross lesions included severe lung consolidation with fibrinous exudate on the pleura and air sacs, petechiae on the epicardium, and increased cloudy fluid in the pericardial sac . Liver and spleen were moderately enlarged . Histologically, there was severe fibrinoheterophilic inflammation in the airways, pleura, and air sacs and severe perivascular interstitial edema in the lungs . Liver had acute coagulative necrosis of hepatocytes associated with occasional thrombosis at the periphery of the liver lobes . Ornithobacterium rhinotracheale was isolated from tissues of the respiratory system, such as infraorbital sinus, trachea, lung, and air sacs, but not from the liver, spleen, or bone marrow . Escherichia coli was isolated less often from lung, air sac, and trachea.

Avian Dis, 1996 Oct-Dec, 40(4), 837 - 40
The production of colibacillosis in turkeys following sequential exposure to Newcastle disease virus or Bordetella avium, avirulent hemorrhagic enteritis virus, and Escherichia coli; Pierson FW et al.; Female large white turkeys were intranasally inoculated with either Newcastle disease virus (ND) or Bordetella avium (BA) at 4 weeks of age . This was followed by oral inoculation with an avirulent (vaccine) strain of hemorrhagic enteritis virus (HE) at 5 weeks and intravenous inoculation with Escherichia coli (EC) at 6 weeks . Control birds received ND, BA, or HE followed by EC; EC alone; or nothing at all . Turkeys receiving one agent prior to EC challenge did not experience a significant increase in mortality or pericarditis . Those exposed to ND or BA followed by HE and EC experienced a significant elevation in mortality and pericarditis . A highly significant positive correlation between the number of infectious agents encountered during primary exposure and the incidence of colibacillosis after EC challenge was demonstrated.

Mol Cell Biochem, 1996 Oct-Nov, 163-164, 319 - 27
Significance of the adenine nucleotide translocator in the pathogenesis of viral heart disease; Schultheiss HP et al.; We found recently autoantibodies against the adenine nucleotide translocator (ANT), a carrier in the inner mitochondrial membrane, in sera of patients with myocarditis and dilated cardiomyopathy . To elucidate whether these antibodies are of pathophysiological importance, we investigated the function and expression of the adenine nucleotide translocator (ANT) in the heart muscle tissue of patients suffering from myocarditis and DCM . We found a markedly lowered transport capacity of the translocator accompanied by an elevation in total ANT protein content . The alteration in ANT protein amount is caused by an ANT isoform shift characterized by an increase in ANT 1 isoform protein associated with a decrease in ANT 2 isoform and an unchanged ANT 3 content . It could be shown that the isoform shift is not a progressive process during the disease period but an event in the early period of illness which becomes permanent . Simulating the effect of pathogenetic factors of autoimmunological diseases, we infected A/J mice with the enterovirus Coxsackie B3 and immunized guinea pigs with myocardial ANT protein . Both treatments led to autoimmunological responds and to a lowered myocardial transport capacity of ANT, to a disturbed energy metabolism and consequently to a depression of heart function.

Electrophoresis, 1996 Oct, 17(10), 1564 - 72
A procedure for protein elution from reverse-stained polyarcylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis; Castellanos-Serra LR et al.; We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band) . Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing . Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern . Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g . high performance liquid chromatography (HPLC) and automatic sequencing . A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E . coli proteins . Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band . This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields . For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm . By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.

Electrophoresis, 1996 Oct, 17(10), 1537 - 41
Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids; Hardy E et al.; Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide of agarose gels . After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min . As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands . Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white-stained background . On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc precipitate is removed from the positions were nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background . The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/band for different 22-base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 microgram/band for human peripheral-blood monocyte RNA . After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel . The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes . Using zinc-imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and anti-HBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E . coli XL-1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV-irradiation at 312 nm) to 10,000.

Neurosci Res, 1996 Oct, 26(2), 95 - 107
Genetic dissection of sexual orientation: behavioral, cellular, and molecular approaches in Drosophila melanogaster; Yamamoto D et al.; Insertional mutagenesis using P-element vectors yielded several independent mutations that cause male homosexuality in Drosophila melanogaster . Subsequent analyses revealed that all of these insertions were located at the same chromosomal division, 91B, where one of the inversion breakpoints responsible for the bisexual phenotype of the fruitless (fru) mutant has been mapped . In addition to the altered sexual orientation, the fru mutants displayed a range of defects in the formation of a male-specific muscle, the muscle of Lawrence . Since the male-specific formation of this muscle was dependent solely on the sex of the innervating nerve and not on the sex of the muscle itself, the primary site of action of the fru gene should be in the neural cells . satori, one of the P-insertion alleles of fru which we isolated, carried the lacZ gene of E . coli as a reporter, and beta-galactosidase expression was found in a subset of brain cells including those in the antennal lobe in the satori mutant . Targeted expression of a sex determination gene, transformer (tra), was used to produce chromosomally male flies with certain feminized glomeruli in the antennal lobe . Such sexually mosaic flies courted not only females but also males when the DM2, DA3 and DA4 glomeruli were feminized, indicating that these substructures in the antennal lobe may be involved in the determination of the sexual orientation of flies . Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts, one of which encodes a putative transcription regulator with a BTB domain and two zinc finger motifs . In the 5' non-coding region, three putative Transformer binding sites were identified . It appears plausible therefore that the fru gene is one of the elements in the sex determination cascade that controls sexual fates of certain neuronal cells . Improper sex determination in these neural cells may lead to altered sexual orientation and malformation of the male-specific muscle . Some implications of the results of our study on sexual orientation in other organisms will be discussed based on the Drosophila research.

J Biomol NMR, 1996 Oct, 8(3), 252 - 60
Selectively 13C-enriched DNA: evidence from 13C1' relaxation rate measurements of an internal dynamics sequence effect in the lac operator; Paquet F et al.; In order to study some internal dynamic processes of the lac operator sequence, the 13C-labeled duplex 5'd(C0G1C2T3C4A5C6A7A8T9T10).d(A10A9T8T7G6T5G4A3G2C1G0)3' was used . The spreading of both the 1H1' and 13C1' resonances brought about an excellent dispersion of the 1H1'-13C1' correlations . The spin-lattice relaxation parameters R(Cz), R(Cx,y) and R(Hz --> Cz) were measured for each residue of the two complementary strands, except for the 3'-terminal residues which were not labeled . Variation of the relaxation rates was found along the sequence . These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo {(1982) J . Am . Chem . Soc., 104, 4546-4570} and extended to three parameters by Clore et al . {(1990) Biochemistry, 29, 7387-7401; and (1990) J . Am . Chem . Soc., 112, 4989-4991} . A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function . With this approach, the global correlation time was found to be the same for each residue . All the C1'-H1' fragments exhibited both slow (tau s = 1.5 ns) and fast (tau f = 20 ps) restricted libration motions (Ss2 = 0.74 to 1.0 and Sf2 = 0.52 to 0.96) . Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex . The possible role played by the special structure of the AATT sequence is discussed . No evident correlation was found between the amplitude motions of the complementary residues . The 5'-terminal residues showed large internal motions (S2 = 0.5), which describe the fraying of the double helix . Global examination of the microdynamical parameters Sf2 and Ss2 along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (Sf2 = 0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin . Moreover, the fit of both R(Cz) and R(Hz --> Cz) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(Cx,y) for the purine residues in the (5' --> 3') G4A3 and A10A9T8T7 sequences . This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale . It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator {Boelens et al . (1987) J . Mol . Biol., 193, 213-216} and that these motions could be implied in the lac-operator-lac-repressor recognition process.

Prostaglandins Leukot Essent Fatty Acids, 1996 Oct, 55(4), 269 - 77
Cloning, sequencing and expression of a 5-lipoxygenase from Syrian hamster embryo fibroblasts; Kitzler JW et al.; The hamster ortholog of human and rat 5-lipoxygenase (5-LO) was cloned from a Syrian hamster embryo (SHE) cell line . A combination of polymerase chain reaction (PCR) and 5' and 3' RACE (rapid amplification of cDNA ends) was used to isolate the complete cDNA for this gene . The cDNA sequence demonstrates the extreme sequence conservation found in this gene family, with a deduced amino acid sequence 95% identical to the rat 5-LO, and 90% identical to the human enzyme . The hamster 5-LO was expressed in E . coli . The expressed protein was detected by an antibody to human 5-LO, and had an apparent molecular weight of 75-80 kD . The products of the action of this enzyme on arachidonic acid are 5-HETE and the diHETEs resulting from the breakdown of LTA4, in a pattern similar to that produced by the recombinant human 5-LO . No oxidation of linoleic acid by this enzyme was detected.

J Chem Neuroanat, 1996 Oct, 11(4), 243 - 56
Nitric oxide synthase-containing magnocellular neurons of the rat hypothalamus synthesize oxytocin and vasopressin and express Fos following stress stimuli; Hatakeyama S et al.; We investigated the chemical and anatomical features of nitric oxide synthase (NOS)-containing neurons in the paraventricular and supraoptic nuclei in the rat hypothalamus using combinations of enzyme histochemistry, in situ hybridization and immuno-histochemistry . Neurons expressing NOS mRNA completely overlapped with NADPH-diaphorase-positive neurons . Topographical distribution of NOS was segregated from that of CRF-containing parvicellular neurons in the posterior paraventricular nucleus but overlapped with that of magnocellular neurons . In the paraventricular nucleus, 70% of oxytocin neurons contained NOS, which corresponded to one half of NOS neurons . About one third of vasopressin-immunoreactive neurons were NADPH-diaphorase-positive and the same proportion of NADPH-diaphorase-positive neurons were vasopressin-immunoreactive . In the supraoptic nucleus, 50% of oxytocin neurons were NADPH-diaphorase-positive, which corresponded to 40% of NOS neurons . About 25% of vasopressin neurons were NADPH-diaphorase-positive, and 30% of NADPH-diaphorase-positive neurons were vasopressin-immunoreactive . When NADPH-diaphorase histochemistry was performed first, subsequent immunostaining was markedly perturbed . Using fluoro-gold as a retrograde tracer, 4% of NADPH-diaphorase-positive neurons were shown to contribute to the descending projection to the spinal cord . About 40%-50% of NADPH-diaphorase-positive neurons exhibited Fos immunoreactivity after injection of lipopolysaccharide or hypertonic saline, while only 10%-15% of these neurons expressed Fos in response to immobilization or pain . Endogenous NO may be involved in the regulation of magnocellular functions, especially when the internal environment is disturbed.

J Bioenerg Biomembr, 1996 Oct, 28(5), 415 - 20
The coupling of the relative movement of the a and c subunits of the F0 to the conformational changes in the F1-ATPase; Howitt SM et al.; F0F1-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F0F1-ATPase . Because of the subunit stoichiometry and the involvement of both a- and c-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits . Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.

J Bioenerg Biomembr, 1996 Oct, 28(5), 409 - 14
Conformational transmission in ATP synthase during catalysis: search for large structural changes; Futai M et al.; Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits . Defective mutation at beta Gly-149 was suppressed by the second mutations at the outer surface of the beta subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission . Extensive mutant/pseudorevertant studies revealed that beta/alpha and beta/gamma subunits interactions are important for the energy coupling between catalysis and H+ translocation . In addition, long range interaction between amino and carboxyl terminal regions of the gamma subunit has a critical role(s) for energy coupling . These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.

J Bioenerg Biomembr, 1996 Oct, 28(5), 397 - 401
Structural changes in the gamma and epsilon subunits of the Escherichia coli F1F0-type ATPase during energy coupling; Capaldi RA et al.; Structural changes in the Escherichia coli ATP synthase (ECF1F0) occur as part of catalysis, cooperativity and energy coupling within the complex . The gamma and epsilon subunits, two major components of the stalk that links the F1 and F0 parts, are intimately involved in conformational coupling that links catalytic site events in the F1 part with proton pumping through the membrane embedded F0 section . Movements of the gamma subunit have been observed by electron microscopy, and by cross-linking and fluorescence studies in which reagents are bound to Cys residues introduced at selected sites by mutagenesis . Conformational changes and shifts of the epsilon subunit related to changes in nucleotide occupancy sites have been followed by similar approaches.

Genet Anal, 1996 Oct, 13(4), 105 - 11
A biotinylated MutS fusion protein and its use in a rapid mutation screening technique; Geschwind DH et al.; The Escherichia coli DNA mismatch repair protein, MutS, binds single base pair mismatches and short deletions in vivo and in vitro . To adapt this protein for mutation detection, a fusion protein of E . coli MutS with a biotinylated peptide domain has been constructed (MutSb) . The biotinylation tag facilitates MutS detection and binding by avidin without significantly altering the DNA mismatch binding properties of MutS in vitro . We describe a novel and rapid mutation detection method with MutSb using streptavidin-coated magnetic beads and demonstrate that MutSb can also be used to remove mismatch containing DNA fragments from a mixture of DNA fragments in solution.

J Biochem (Tokyo), 1996 Oct, 120(4), 838 - 44
Cloning, expression and characterization of plasma platelet-activating factor-acetylhydrolase from guinea pig; Karasawa K et al.; In a previous study, we purified PAF-acetylhydrolase, which converts PAF to an inactive metabolite, lysoPAF, from peritoneal fluid of guinea pigs subjected to experimental endotoxin shock and found that this purified enzyme had similar biochemical properties to the plasma enzyme {Karasawa, K., Yato, M., Setaka, M., and Nojima, S . (1994) J . Biochem . 116, 374-379} . In this study, we isolated a homogeneous enzyme preparation from guinea pig plasma using a similar procedure . The molecular mass of this purified enzyme, as determined by SDS-PAGE was 58-63 kDa, larger than that (43 kDa) of the human enzyme . To elucidate the molecular structure of this enzyme and clarify its relationships with PAF-acetylhydrolases of other species, we isolated and sequenced a cDNA encoding this enzyme . Its cDNA contains an open reading frame encoding 436 amino acids and its predicted molecular mass (49 kDa) is lower than that of the native enzyme, suggesting that guinea pig plasma PAF-acetylhydrolase, unlike the human enzyme, is modified post-translationally, perhaps by glycosylation.

J Biochem (Tokyo), 1996 Oct, 120(4), 828 - 33
In situ topology of cytochrome b5 in the endoplasmic reticulum membrane; Kuroda R et al.; Cytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions {Mitoma, J . and Ito, A . (1992) EMBO J . 11, 4197-4203} . In situ topology of cytochrome b5 in the ER-membrane was studied using immunofluorescence microscopy . Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome . Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O . The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen . In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O . The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side . Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method . Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.

J Biochem (Tokyo), 1996 Oct, 120(4), 766 - 72
Replication protein-A mediates the association of calf thymus DNA polymerase alpha-DNA primase complex with guanine-rich DNA sequence; Suzuki M et al.; We have shown that calf thymus DNA polymerase alpha-DNA primase complex (pol alpha-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis {Suzuki, M . et al . (1993) Biochemistry 32, 12782-12792} . Here we tested the association of pol alpha-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication . While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with pol alpha-primase using anti-pol alpha antibody, SVG was hardly precipitated under the same conditions . Competition studies in either gel-retardation assay or during de novo DNA synthesis by pol alpha-primase demonstrated that the interaction of pol alpha-primase with SVG was much weaker than that with CTPPS-1 . On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1 . After preincubation with RP-A, SVG could bind pol alpha-primase that was immobilized on Sepharose beads . The simian virus 40 large T antigen also enhanced association of SVG to pol alpha-primase, while Escherichia coli single-stranded DNA-binding protein did not . However, pol alpha-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers . When SVG was extended 10 nucleotides at its 5'-end, pol alpha-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A . These results suggest a new role for RP-A, i.e., as a molecular tether that allows pol alpha-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.

Immunology, 1996 Oct, 89(2), 289 - 94
Biosynthesis of human ficolin, an Escherichia coli-binding protein, by monocytes: comparison with the synthesis of two macrophage-specific proteins, C1q and the mannose receptor; Lu J et al.; Ficolin is characterized by the presence of both collagen-like and fibrinogen-like sequences, and potentially has a similar overall structure as the complement protein C1q and the collectins . Previous studies have reported the presence of human ficolin mRNA predominantly in peripheral blood leucocytes . In the present study, the cellular origin of human ficolin was investigated in further detail . Preliminary studies using reverse transcriptase-polymerase chain reaction (RT-PCR) showed that ficolin mRNA was synthesized by U937 cells, a human monocyte cell line . This finding suggested that blood monocytes also normally synthesize human ficolin . Peripheral blood monocytes from adult human donors were harvested at serial time-points (0-20 hr) after adhesion to tissue culture plates, and total RNA was isolated and assayed for ficolin mRNA by RT-PCR . Ficolin mRNA was highly expressed in monocytes throughout the first 20 hr of adhesion . In contrast, C1q and mannose receptor mRNA were not detectable during the first 8 hr of adhesion, but were highly expressed by 20 hr . Cells were harvested at longer time intervals (1, 2, 4, 6 and 8 days) to determine whether ficolin expression was temporally regulated at later stages of monocyte differentiation . Ficolin mRNA levels decreased sharply from day 1 to day 6 . In contrast, the levels of both C1q and mannose receptor mRNA showed no changing trend . These results are consistent with the absence of ficolin expression in many macrophage-rich tissues previously reported . The origin of ficolin from monocytes, together with its structural similarity to C1q and the collectins, raises the possibility that ficolin may be another plasma protein capable of binding to surface structures of micro-organisms . Escherichia coli was therefore incubated with human serum, and bound proteins, after elution with sugars, were analysed by Western blotting using an antiserum raised against a synthetic ficolin peptide . The antiserum identified a polypeptide of approximately 42000 MW, which is similar in size to that of ficolin as predicted from its cDNA-derived sequence.

Pharmazie, 1996 Oct, 51(10), 753 - 5
Effect of some organic ammonium salts and amine oxides in the SOS chromotest; Majtan V et al.; The effect of two organic ammonium salts, (1-methyldodecyl)trimethylammonium bromide (ATDBr) and tetramethylammonium bromide (TMABr), as well as that of two amine oxides, (1-methyl-dodecyl)dimethylamine oxide (ATDNO) and trimethylamine oxide (TMANO) on the induction of the SOS system in Escherichia coli PQ 37 (SOS chromotest) was examined . The compound with the long alkyl chain in the molecule (ATDBr) showed genotoxic activity in all concentrations tested . In the second group of the amphiphilic compounds tested, amine oxides, no effect of the length of the alkyl chain upon the studied activity was ascertained.

Curr Opin Biotechnol, 1996 Oct, 7(5), 541 - 6
Protein expression using ubiquitin fusion and cleavage; Baker RT; Expressing proteins and polypeptides as fusions to ubiquitin offers the advantage of an often dramatic increase in yield, and the ability to produce any desired amino-terminal residue upon ubiquitin cleavage . The recent availability of cloned ubiquitin-cleaving enzymes has enhanced this technique for both bacterial and eukaryotic host systems.

Curr Biol, 1996 Oct 1, 6(10), 1230 - 3
DNA repair: how yeast repairs radical damage; Cunningham RP; Cloning of the OGG1 gene from Saccharomyces cerevisiae has revealed that DNA glycosylases are not necessarily conserved throughout phylogeny, yet there is a DNA-repair protein superfamily with a wide substrate specificity found from bacteria to man.

Scand J Urol Nephrol, 1996 Oct, 30(5), 425 - 8
Emphysematous cystitis after orchiectomy; Tsiftsis DD et al.; A 59-year-old man developed emphysematous cystitis 3 weeks after undergoing left orchiectomy because of suppurative epididymitis . The case is presented because of its unusual cause and to emphasize the high degree of suspicion required for the diagnosis.

Acta Anaesthesiol Scand, 1996 Oct, 40(9), 1154 - 60
Surfactant treatment in experimental Escherichia coli pneumonia; Song GW et al.; BACKGROUND: Deterioration of lung function in bacterial pneumonia may in part be due to inactivation of endogenous surfactant . We investigated the effects of surfactant treatment on gas exchange and lung morphology in an experimental model of pneumonia caused by Escherichia coli . METHODS: A total of 117 adult rats received via the trachea 2 ml/kg body weight of a standard suspension of Escherichia coli (4 x 10(9) bacteria/ml) . After 2-3 days, 31 of the infected animals showed symptoms of respiratory failure with PaO2 < 27 kPa during ventilation with 100% O2 . All these animals were kept in a multi-plethysmograph system and ventilated for 45 min with a tidal volume of 6 ml/kg, a frequency of 30/min, an inspiration/expiration ratio of 1:1, and a positive end-expiratory pressure of 0.2 kPa . After 15 min of mechanical ventilation, animals were divided in three treatment groups, receiving via the airways (1) no material, (2) normal saline (2 ml/kg), or (3) Curosurf, 80 mg/ml (2 ml/kg) . Ten healthy animals served as controls . Lung-thorax compliance and blood gases were measured 15 and 30 min after surfactant treatment . After the period of ventilation, animals were killed, and the left lung was weighed and fixed in formalin for histological examination . The right lung was washed in situ with normal saline via the tracheal tube . Total phospholipids, and levels of phosphatidylcholine (PC) and protein in lavage fluid were determined . RESULTS: In comparison with pre-treatment values, average PaO2 at 30 min was increased by 76% in animals receiving Curosurf (P < 0.01), but did not improve in the other groups . The left lung weight/body weight ratio showed a nearly 3-fold increase in infected animals in comparison with normal controls . There was also a 3-fold increase in the protein content of lung lavage fluid from infected rats, but values for total phospholipids and PC content were unchanged in animals not receiving surfactant . Histological examination of the lungs showed wide-spread non-specific pneumonia in infected animals, but no difference in alveolar air expansion between surfactant-treated and non-treated ones . CONCLUSION: Surfactant replacement significantly improves oxygenation in rats with E . coli pneumonia, without affecting lung-thorax compliance during mechanical ventilation or alveolar expansion pattern in lungs fixed by conventional methods.

Protein Eng, 1996 Oct, 9(10), 857 - 67
Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI; Kashiwagi T et al.; Three mutants of Escherichia coli ribonuclease HI, in which an invariant acidic residue Asp134 was replaced, were crystallized, and their three-dimensional structures were determined by X-ray crystallography . The D134A mutant is completely inactive, whereas the other two mutants, D134H and D134N, retain 59 and 90% activities relative to the wild-type, respectively . The overall structures of these three mutant proteins are identical with that of the wild-type enzyme, except for local conformational changes of the flexible loops . The ribonuclease H family has a common active site, which is composed of four invariant acidic residues (Asp10, Glu48, Asp70 and Asp134 in E.coli ribonuclease HI), and their relative positions in the mutants, even including the side-chain atoms, are almost the same as those in the wild-type . The positions of the delta-polar atoms at residue 134 in the wild-type, as well as D134H and D134N, coincide well with each other . They are located near the imidazole side chain of His124, which is assumed to participate in the catalytic reaction, in addition to the four invariant acidic residues . Combined with the pH profiles of the enzymatic activities of the two other mutants, H124A and H124A/D134N, the crystallographic results allow us to propose a new catalytic mechanism of ribonuclease H, which includes the roles for Asp134 and His124.

Protein Eng, 1996 Oct, 9(10), 833 - 42
Local structural motifs of protein backbones are classified by self-organizing neural networks; Schuchhardt J et al.; Important and relevant information is expected to be encoded in local structural elements of proteins . An unsupervised learning algorithm (Kohonen algorithm) was applied to the representation and unbiased classification of local backbone structures contained in a set of proteins . Training yielded a two-dimensional Kohonen feature map with 100 different structural motifs including certain helical and strand structures . All motifs were represented in a phi-psi-plot and some of them as a three-dimensional model . The course of structural motifs along the backbone of four selected proteins (cytochrome b5, cytochrome b562, lysozyme, gamma crystallin) was investigated in detail . Trajectories and histograms visualizing the abundance of characteristic motifs allowed for the distinction between different types of protein overall folds . It is demonstrated how the histograms may be used to construct a structural similarity matrix for proteins . The Kohonen algorithm provides a simple procedure for classification of local protein structures independent of any a priori knowledge of leading structural motifs . Training of the Kohonen network leads to the generation of "consensus structures' serving for the task of classification.

Mol Microbiol, 1996 Oct, 22(2), 379 - 88
The N-terminal beta-barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization; Mol O et al.; The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer . The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones . FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones . A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues . Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis . Two hybrid chaperones were constructed . Each hybrid protein contained one beta-barrel domain of FaeE and the other beta-barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively) . Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae . The Fan/FaeE hybrid chaperone was active in the bio-synthesis of K99 fimbriae . The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG . Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone . Altogether, the data suggested that the N-terminal beta-barrel domain of a fimbrial chaperone determines subunit specificity . In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.

Mol Microbiol, 1996 Oct, 22(2), 339 - 55
Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader; Wu CJ et al.; The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology . The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon . Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E . coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation . Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence . Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E . coli and S . lividans.

Mol Microbiol, 1996 Oct, 22(2), 275 - 82
Co-ordination between membrane oriC sequestration factors and a chromosome partitioning protein, TolC (MukA); Bahloul A et al.; oriC DNA in the hemimethylated (but not in the fully methylated) state reacts with an Escherichia coli K-12 outer membrane preparation . This reaction is drastically reduced when the membrane preparation of a seqA null mutant is used . An in vitro reconstitution of the activity was undertaken by adding a partially purified SeqA protein to a seqA mutant membrane without success . A possible reason for this failure might be a profound modification of the outer membrane of the seqA mutant (as revealed by the fact that membrane from the mutant sediments more slowly than that from the wild type during ultracentrifugation) . There is also a reduction in the content of OmpF protein . Moreover, one of the minor outer membrane proteins involved in partitioning of newly synthesized chromosomes, the ToiC (MukA) protein, was also found to be downregulated in the seqA mutant . This is also true of the hobH mutant grown in a high-osmolarity medium . Mutants of both seqA and hobH stop dividing after hyperosmotic shock, forming filaments (as observed in dam mutants).

Mol Microbiol, 1996 Oct, 22(2), 231 - 7
FtsZ-spirals and -arcs determine the shape of the invaginating septa in some mutants of Escherichia coli; Addinall SG et al.; The essential cell division protein FtsZ forms a dynamic ring structure at the future division site . This Z-ring contracts during cell division while maintaining a position at the leading edge of the invaginating septum . Using immunofluorescence microscopy we have characterized two situations in which non-ring FtsZ structures are formed . In ftsZ26 (temperature sensitive, Ts) mutant cells, FtsZ-spirals are formed and lead to formation of spirally invaginating septa, which in turn cause morphological abnormalities . In rodAoul mutant cells, which grow as spheres instead of rods, FtsZ-arcs are formed where asymmetric septal invaginations are initiated . The FtsZ-arcs later mature into complete FtsZ-rings . Our data show that Z-spirals and Z-arcs can contract and that in doing so, they determine the shape of the invaginating septa . These results also strongly suggest that in normal cell division, FtsZ is positioned to a single nucleation site on the inner membrane, from which it polymerizes bidirectionally around the cell circumference to form the Z-ring.

Mol Microbiol, 1996 Oct, 22(2), 197 - 205
Construction of derivatives of the F plasmid pOX-tra715: characterization of traY and traD mutants that can be complemented in trans; Maneewannakul K et al.; A derivative of the F plasmid, pOX38-tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7 . A series of plasmids related to pOX38-tra715 were constructed which carry either deletion mutations or point mutations in traY . When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied In trans from compatible plasmids . Insertion of a kanamycin-resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild-type PY promoter, resulted in loss of F piliation and transfer ability . Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter, pOX38-tra719-traD411, which contains a chloramphenicol-resistance cassette in place of the kanamycin-resistance cassette in pOX38-tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.

Eur J Gastroenterol Hepatol, 1996 Oct, 8(10), 1021 - 2
Unusual presentation of a tuberculous peritonitis in a patient with concomitant AIDS and liver cirrhosis; Turner L et al.; We report a case of tuberculous peritonitis in a patient with concomitant HIV infection and liver cirrhosis . A 50-year-old man with viral B and delta liver cirrhosis and AIDS was diagnosed with spontaneous Escherichia coli peritonitis and successfully treated with beta-lactamins . Three months later, ascites reappeared and Mycobacterium tuberculosis was identified in peritoneal fluid cultures . The triple antituberculosis regimen was adjusted to his level of liver failure but the patient died of hepatic encephalopathy . Concomitant HIV infection and liver cirrhosis favour tuberculous peritonitis but they also make its diagnosis extremely difficult . Considering the poor prognosis of this infection when untreated, tuberculous peritonitis should be systematically suspected in such patients.

Infect Immun, 1996 Oct, 64(10), 4027 - 34
Repetitive elements of the Mycoplasma hominis adhesin p50 can be differentiated by monoclonal antibodies; Henrich B et al.; The gene encoding p50, an adhesin of Mycoplasma hominis, was identified, cloned, and sequenced . Comparison of the derived amino acid sequence with the N-terminal amino acids sequenced by the Edman reaction of the native protein revealed that p50 is expressed as a 467-amino-acid precursor . Posttranslational modification leads to a 441-amino-acid lipoprotein with an extended, predominantly helical structure and a leucine zipper . Computer analysis of the amino acid sequence identified a threefold-repetitive sequence motif comprising approximately three-quarters of the total protein . Different regions of the p50 polypeptide chain were expressed in Escherichia coli . Western blot (immunoblot) analysis of the E . coli lysates revealed that the epitopes of four p50-specific monoclonal antibodies were localized in the middle and C-terminal part of the protein . Epitope mapping by exonuclease III digestion showed that all of the four monoclonal antibodies bound within the same region of the threefold-repetitive amino acid sequence motif . The repeats, which were highly homologous but not identical in structure, could be differentiated by the monoclonal antibodies.

Biol Chem, 1996 Oct, 377(10), 625 - 32
The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV; Wende W et al.; A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described . It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes . Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme . The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag . The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography . Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits . To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced . While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA . From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.

Anticancer Drug Des, 1996 Oct, 11(7), 553 - 67
Synthesis and evaluation of 4-substituted analogues of 5-{N,N-bis (2-chloroethyl)amino}-2-nitrobenzamide as bioreductively activated prodrugs using an Escherichia coli nitroreductase; Atwell GJ et al.; 2,4-Dinitrobenzamide mustards, exemplified by the parent compound SN 23862 (2) are activated under aerobic conditions by an Escherichia coli nitroreductase enzyme (NR2) via selective reduction of the 2-nitro group, and are thus of interest as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT) . A series of related compounds 12a-12d, where the 4-nitro group of 2 was replaced by other substituents of varying electronic properties, were prepared and evaluated as potential ADEPT prodrugs . One-electron reduction potentials of the compounds correlated well with the substituent sigma m values, with the exception of the unsubstituted (4-H) analogue 13, which had a much lower value than expected on electronic grounds, due to a coplanar conformation of the mustard . The cytotoxicities of the compounds towards aerobic UV4 cells correlated positively with the electron-donating ability of the 4-substituent (measured by sigma p values), indicating that the cytotoxicities of the compounds in the absence of the NR2 enzyme are due substantially to the parent (unreduced) compounds . A positive, although less strong, correlation was seen between the electronic properties of the 4-substituent and their cytotoxicities in the presence of the NR2 enzyme, suggesting that, in this closely related series, the degree of activation by the enzyme is significantly dependent on the reduction potential of the 2-nitro group . While the 4-SO2Me derivative 12d was the next most preferred substrate after the parent 2, it was considerably less so (degree of activation as measured by IC50 ratio of 26 compared with 145), despite the similar electronic properties of the two 4-substituents.

Anal Biochem, 1996 Oct 1, 241(1), 30 - 4
A procedure for isolation of pure complexes of acylated and unacylated tRNA(Phe) with ribosomes from Escherichia coli; Chinali G; A simple and rapid procedure for the isolation of pure specific complexes of poly(U).ribosome with acylated and/or unacylated tRNA(Phe) is described . The method is based on the use of conditions that favor the formation of polyribosomes . The polyribosomes are separated from ribosomal subunits and monoribosomes by gel filtration on Sepharose 4B at low Mg2+ concentrations that cause the selective dissociation of tRNA-vacant ribosomes from polyribosomes . The procedure allows the isolation in about one hour of various ribosome.poly(U).tRNA complexes completely free of ribosomal particles unable to bind tRNA . A minor fraction of the purified ribosomal complexes is formed by particles devoid of peptidyltransferase activity . Attempts to eliminate this fraction of inactive ribosomes and to obtain ribosomal complexes fully active in polypeptide synthesis were unsuccessful.

Anal Biochem, 1996 Oct 1, 241(1), 23 - 9
Detection and purification of sequence-specific DNA binding protein; Majumdar KC et al.; A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane using a slot-blot apparatus, incubating with 32P-labeled DNA probe in buffer in the presence of excess of nonspecific E . coli DNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal . A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), designated as Bkm-binding protein (BBP), is also described . This requires only a DNA affinity column after the initial ammonium sulfate precipitation . The insert (545 bp) of the Drosophila clone 2(8) containing 66 copies of GATA repeats was used to prepare the sequence-specific DNA-Sepharose affinity column . The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins in general.

Nephrol Dial Transplant, 1996 Oct, 11(10), 1983 - 8
Identification of post-transplant anti-alpha 5 (IV) collagen alloantibodies in X-linked Alport syndrome; Dehan P et al.; X-linked Alport syndrome (AS) is a heritable disorder which is associated with mutations in the type IV collagen alpha 5 (IV) chain gene (COL4A5) located on chromosome X . Following renal transplantation, an average of 6% of male AS patients develop anti-GBM nephritis . We studied the specificity of the antibodies against type IV collagen in the serum of a patient with COL4A5 partial deletion . The specificity of these alloantibodies was determined against collagenase-digested GBM, as well as against recombinant non-collagenous (NC1) domains of the type IV collagen alpha 1(IV)-alpha 6(IV) chains expressed in escherichia coli . Immunoblotting and ELISA demonstrated that these antibodies bound specifically to the NC1 domain of alpha 5(IV) collagen . There was no binding to the NC1 domain of the other chains, including the Goodpasture antigen . Competitive ELISA confirmed the results obtained by ELISA and immunoblotting . This patient developed alloantibodies directed against antigens present in the grafted kidney, but absent from his Alport kidney . The pathogenesis of post-transplantation glomerulonephritis in the Alport patient studied is thus similar to that of Goodpasture syndrome, with the exception that the pathogenic antibodies are targeted to another alpha chain of type IV collagen.

FEMS Microbiol Rev, 1996 Oct, 19(1), 25 - 52
Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli; Mol O et al.; Fimbriae are long filamentous polymeric protein structures located at the surface of bacterial cells . They enable the bacteria to bind to specific receptor structures and thereby to colonise specific surfaces . Fimbriae consist of so-called major and minor subunits, which form, in a specific order, the fimbrial structure . In this review emphasis is put on the genetic organisation, regulation and especially on the biosynthesis of fimbriae of enterotoxigenic Escherichia coli strains, and more in particular on K88 and related fimbriae, with ample reference to well-studied P and type 1 fimbriae . The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher ('doorkeeper') . Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the (ordered) translocation of the subunits across the outer membrane and their assembly into a growing fimbrial structure will be described . A model for K88 fimbriae is presented.

J Med Virol, 1996 Oct, 50(2), 159 - 67
Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C; Fournillier-Jacob A et al.; Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease . A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40 . As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used . Of 38 chronically infected patients positive for anti-E2 antibodies by IFA, 26 were positive in the Western blot assay (68%) and 25 in the INNO-LIA test (66%) . Thus, the detection of anti-envelope antibodies is highly dependent on the antigen formulation, and a native glycosylated form of the proteins is probably needed for their efficient detection . This study shows that the antibody response to HCV envelope proteins depends on the phase of infection . A few acutely infected patients displayed a response to E1 or E2 (36% by Western blot, 7% by IFA), and these antibodies seem to develop in patients evolving toward chronicity . The high prevalence in chronically infected subjects (62% to E2 by Western blot, 90% by IFA), particularly in subjects with essential mixed cryoglobulinemia (68% and 100%), confirms that the resolution of infection involves more than these antibodies . The antienvelope response in patients treated with interferon was investigated, but no significant relationship was found between antibody level prior to treatment and the evolution of hepatitis . The detection of anti-envelope antibodies, therefore, is not predictive of the response to antiviral therapy.

Mol Reprod Dev, 1996 Oct, 45(2), 107 - 16
Cloning and expression of recombinant rabbit fertilin; Hardy CM et al.; Fertilin is a sperm surface protein complex which is reported to play an essential role in sperm-egg fusion in mammals . It is comprised of two related subunits, alpha and beta, both of which are glycosylated and have cytoplasmic and extracellular domains . This protein has been reported to play an essential role in sperm-egg fusion in mammals . We report on the cloning and sequencing of the complete cDNA sequences of both subunits from rabbit testis, and the production of recombinant proteins for testing their potential as antigens for use in an immunocontraceptive vaccine to control wild rabbit populations . The cDNAs for rabbit fertilin alpha and beta (Genbank accession numbers, U46069 and U46070) are predicted to encode proteins of 919 and 751 amino acids, respectively, and to show significant levels of homology to fertilin subunits isolated from other species . Analysis of the predicted protein sequences of fertilin alpha but not beta reveals the presence of 21 direct repeats of the hexameric sequence A/PPPPEA at the extreme carboxy terminus, similar to what has been described for a fertilin alpha gene isoform in the monkey . DNA sequences corresponding to the predicted mature alpha and beta fertilin subunits were individually cloned into a bacterial expression system, and the recombinant proteins were used to raise polyclonal antibodies in mice . These antibodies detect components of the native fertilin complex from rabbit sperm.

Hybridoma, 1996 Oct, 15(5), 333 - 42
Identification of functional domains on gC1Q-R, a cell surface protein that binds to the globular "heads" of C1Q, using monoclonal antibodies and synthetic peptides; Ghebrehiwet B et al.; A membrane protein (33 kDa) that binds to the globular "heads" of C1q (gC1q-R) has been recently described . The full length cDNA encoding gC1q-R has been cloned, expressed in E . coli and using the purified recombinant protein (rgC1q-R) as an immunogen, a panel of IgG monoclonal antibodies (MAb) has been produced by fusion of spleen cells from hyperimmunized BALB/c mice with NSO mouse myeloma partners . From this fusion, 60 anti-gC1q-R hybridomas were selected and evaluated for their ability to (1) discriminate between the mature form (MF) of gC1q-R (residues 74-282) and a truncated form (TF) lacking residues 74-95, which contains a major C1q binding site, (2) recognize two functionally defined synthetic peptides derived from the NH2-(XN18) and COOH-(XC15) terminus of gC1q-R, and (3) bind to microtiter well fixed intact Raji cells . Several clones were identified: MAbs 46.23 and 60.11 (IgG1 kappa), reacted strongly with ELISA plate-fixed intact Raji and K562 cells, MF, and the XN18 peptide, but had poor or no reactivity with TF; MAbs 74.5.2 > 25.15 (IgG1 kappa) recognized both MF and TF and are directed against epitopes in the XC15 peptide that contains a binding site for high-molecular-weight kininogen and Factor XII.

Biol Pharm Bull, 1996 Oct, 19(10), 1387 - 9
Molecular cloning and functional expression of cDNAs for Glycyrrhiza glabra squalene synthase; Hayashi H et al.; We have isolated two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase of Glycyrrhiza glabra L . by cross-hybridization with that of Arabidopsis thaliana squalene synthase under conditions of low stringency . Their nucleotide sequences contained an open reading frame for a polypeptide of 413 amino acids (GgSQS1) and 412 amino acids (GgSQS2), respectively . The deduced amino acid sequence of GgSQS1 exhibits 88%, 81%, 78%, 45-44%, and 45-41% identity to those of the GgSQS2, Nicotiana benthamiana . Arabidopsis thaliana, mammal, and yeast squalene synthases, respectively . To express the G . glabra enzymes in Escherichia coli, the entire coding region was subcloned into an expression vector . The cell-free extracts of E.coli transformed with the respective plasmid converted 3H-farnesyl diphosphate into squalene.

Biol Pharm Bull, 1996 Oct, 19(10), 1275 - 8
Growth phenotypes of Escherichia coli carrying a mutation of acidic phospholipid synthesis; Akimitsu N et al.; We identified novel phenotypes of a pgsA3 mutant lacking the potential to synthesize phosphatidylglycerophosphate, a precursor of phosphatidylglycerol . The first phenotype is limited cell growth at a high temperature, under the condition of low salt . The phenotype was co-transduced with a phenotype lacking the potential to synthesize phosphatidylglycerol in a P1 transduction experiment, and was restored by transformation with a plasmid containing a wild type pgsA gene . The second phenotype of the pgsA3 mutant was resistant to growth in the presence of a low concentration of kanamycin (4 micrograms/ml) . P1 transduction and transformation with the plasmid containing the wild-type pgsA gene revealed that the pgsA3 mutation was also responsible for the second phenotype.

Biol Pharm Bull, 1996 Oct, 19(10), 1271 - 4
Antibody-producing effects in mice by synthetic immunoactive lipopeptides with the conjugated amino acid sequence of gp120 in human immunodeficiency virus; Shimizu T et al.; To induce peptide-specific antibodies in mice, as a model for vaccination against human immunodeficiency virus (HIV), lipopeptide analogs conjugated with three repeating units (KAB-112; designated as gp120-peptide) of a part (Gly-Pro-Gly-Arg-Ala-Phe) of the amino acid sequences of the V3 loop region in gp120 of HIV were synthesized . The mitogenicity, production of nitric oxide (NO) and induction of peptide-specific antibodies in mice by synthetic lipopeptides were examined . Compounds, KAB-8 (diacylglycerol-tetrapeptide having a part of the amino acid sequence in Escherichia coli), KAB-116 (diacylglycerol-cysteine), KAB-117 (diacylglycerol with gp120-peptide) and KAB-121 (KAB-8 with gp120-peptide) were capable of increasing significantly the incorporation of {3H}thymidine into splenocytes of C3H/He mice at concentrations ranging from 12.5 to 100 microM, but KAB-112 (gp120-peptide) and KAB-115 (monoacylglycerol with gp120-peptide) did not show such activity . The compounds, KAB-8, KAB-117 and 121, exhibited NO production in murine macrophages . When 50 nmol of these compounds was administered intraperitoneally into BALB/c mice on days 0, 16 and 46, the peptide-specific antibody titers in their sera produced by each compound were determined with ELISA . The sera of KAB-117 and KAB-121, which were obtained on days 14, 30, 42, 57 and 70, had a higher titer than that of KAB-112 and KAB-115, suggesting that the diacylglycerol derivative enhance the production of the peptide-specific antibodies.

Biol Pharm Bull, 1996 Oct, 19(10), 1254 - 60
An epitope chimeric antigen for the hepatitis C virus serological screening test; Yagi S et al.; The epitope chimeric antigen, CepCM, composed of the 9 selected major epitope regions (two in NS3, two each in the NS4 of two genotypes, two each in the core of two genotype, and one in the core in hepatitis C virus (HCV) polypeptide), was expressed as a fusion protein of the trpE peptide in E . coli . An ELISA test using this antigen produced the same judgements with most of the panel sera as a second generation HCV screening kit . Though discrepancies were found in twelve samples (5% of the samples), further analysis revealed that eleven samples were indeterminate sera as judged by an immunoblot test . The reactivity found in several seroconversion series sera suggested that CepCM has superior reactivity to HCV infected sera than some second generation kits . These data indicated that an epitope chimeric antigen with a man-made sequence will be a excellent tool for a diagnostic test kit.

Exp Hematol, 1996 Oct, 24(12), 1369 - 76
Molecular cloning and characterization of murine interleukin-11; Morris JC et al.; Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells . In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11 . The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors . The murine IL-11 protein was synthesized as a soluble thioredoxin-IL-11 fusion in Escherichia coli and the expression of murine IL-11 was examined by pulse-chase radiolabeling in COS cells . The chromosomal location of the murine IL-11 gene was assigned to the proximal arm of chromosome 7.

J Interferon Cytokine Res, 1996 Oct, 16(10), 845 - 52
Human interferon-alpha receptor: identification of the region involved in binding to interferon-alpha B; Hirata MH et al.; Three polypeptides comprising amino acids 1-102, 93-260, and 261-410 of the extracellular domain of the human interferon-alpha receptor HuIFN-alpha R (Uze, G., Lutfalla, G., and Gresser, I . Cell 1990; 60:225-234) have been expressed in Escherichia coli . The polypeptides were sequestered within bacterial inclusion bodies . Inclusion body material was solubilized by 8 M urea, and the polypeptides were purified by gel filtration or histidine tag-based affinity chromatography . Overall recovery of each purified and refolded polypeptide was approximately 0.5-0.8 mg/liter of cell culture . The polypeptides migrated as homogeneous monomers of 12 kDa, 22 kDa, and 17 kDa, respectively on reduced sodium dodecylsulfate polyacrylamide gel electrophoresis . The polypeptide fragments corresponding to amino acids 1-102, and 93-260 of the extracellular domain of HuIFN-alpha R lacked the ability to bind to IFN-alpha B and to inhibit its biologic activities . The polypeptide fragment corresponding to amino acids 261-410 of the receptor molecule inhibited the antiproliferative activity of IFN-alpha B and competed with the Daudi cell surface receptor for binding to this IFN-alpha species.

J Interferon Cytokine Res, 1996 Oct, 16(10), 835 - 44
Isolation of a biologically active soluble human interferon-alpha receptor-GST fusion protein expressed in Escherichia coli; Nguyen NY et al.; The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uze, G., Lutfalla, G., and Gresser, I . Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase . The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies . Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100 . The soluble fusion protein was purified by gel filtration and affinity chromatography . Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture . The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis . The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B . We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.

Trends Genet, 1996 Oct, 12(10), 418 - 24
Base-modification mRNA editing through deamination--the good, the bad and the unregulated; Smith HC et al.; RNA editing is a co- or post-transcriptional process in which select nucleotide sequences in RNA are altered from that originally encoded in the genome . The mRNAs encoding apolipoprotein B and some glutamate receptor subunits of ionotropic membrane channels are edited by site-specific base-deamination systems . Although these editing systems differ markedly in their mechanism for RNA-substrate binding and in their catalytic subunits, recent results suggest potentially common solutions to the problem of editing-site selectivity . The data suggest that there are multiple editing complexes or 'editosomes', which manifest editing-site preferences due to their macromolecular composition.

Gene Ther, 1996 Oct, 3(10), 868 - 77
Protection of mammalian cells against chloroethylating agent toxicity by an O6-benzylguanine-resistant mutant of human O6-alkylguanine-DNA alkyltransferase; Hickson I et al.; Low levels of expression in haemopoietic cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (A Tase), is associated with the dose-limiting sensitivity of these cells to the chemotherapeutic chloroethylating and related methylating agents . Thus, the use of agents which deplete ATase such as O6-benzylguanine (O6-beG), as a tumour sensitisation strategy is likely further to potentiate collateral toxicity in bone marrow . In order to address this problem, we have engineered two mutants of human ATase (hAT) for resistance to O6-beG and characterised the in vitro properties of the proteins . In one mutant protein (hATPA), the proline at position 140 was changed to an alanine, whilst in the other (hATPA/GA) an additional mutation (glycine 156 to alanine) was also introduced . The I50 values for O6-beG of hAT, hATPA and hATPA/GA are 0.16, 2.5 and > 500 microM respectively, indicating that hATPA is resistant and hATPA/GA effectively refractory to O6-beG inactivation . Both mutant proteins retain comparable methyl transfer kinetics to those of nonmutant hAT and although they are thermally less stable in vitro than the wild-type protein, both can be substantially stabilised by DNA . Expression of either hAT or hATPA/GA following gene transfer into RJKO cells, raised the D37 value for mitozolomide from 0.35 microgram/ml for control cells to 10 micrograms/ml in the absence of O6-beG . However, whilst hAT-mediated protection was ablated by 20 microM O6-beG, the hATPA/GA protein provided protection against mitozolomide under the same conditions . Similar observations were made with chlorozotocin . The data suggest that transfer and expression of O6-beG resistant ATase in normal progenitor cells, should be a useful therapeutic strategy to protect the cells from the cytotoxic effects of the O6-alkylating agents even when used in combination with tumour sensitising agents such as O6-beG.

Biochem Mol Biol Int, 1996 Oct, 40(3), 469 - 77
Cloning of a cDNA encoding a developmentally regulated 22 kDa polypeptide from tobacco leaf plasma membrane; Gantet P et al.; A polypeptide doublet (P18-P19, ca 22 kDa, pI 4.5) has been shown to accumulate in tobacco leaf plasma membrane in a development-dependent way, under constant environmental conditions . P18 and P19 were purified by 2D-PAGE and microsequenced . Microsequences revealed only small differences between the two polypeptides . A PCR-based cloning strategy identified a cDNA displaying a 591 bp ORF . The encoded polypeptide contained P19 specific microsequences . It was expressed in E . coli and a specific rabbit antiserum was raised . Western-blots confirmed its identification as P19 . The accumulation pattern of hybridizable mRNA around the floral induction period was similar to that of P18 and P19 . Searching of databases revealed no significant hits except unidentified plant ESTs . P18 and P19 are proposed as the first example of plant-specific and developmentally regulated plasma membrane proteins.

J Vet Pharmacol Ther, 1996 Oct, 19(5), 382 - 88
A lipopolysaccharide-induced acute phase response in the pig is associated with a decrease in hepatic cytochrome P450-mediated drug metabolism; Monshouwer M et al.; Drug disposition, including hepatic drug metabolism, is markedly affected by infection, inflammation and other conditions that invoke the acute phase response . In the present study, an Escherichia coli lipopolysaccharide (LPS)-induced acute phase response model was developed in pigs . This model was used to study the effects of the acute phase response on drug disposition and hepatic drug metabolism in vivo and in microsomal preparations . The results obtained were compared with those from Actinobacillus pleuropneumoniae-infected pigs . Intermittent intravenous administration of LPS induced a mild acute phase response as evidenced by increased rectal body temperatures, anorexia and increased cytokine (TNF-alpha and IL-6) serum levels within 1-2 h after the first LPS injection . The acute phase response is associated with a pronounced decrease of antipyrine plasma clearance (control 8.5 +/- 0.8 vs . LPS 2.2 +/- 0.7 mL/min.kg) . Furthermore, total cytochrome P450 content and microsomal cytochrome P450-dependent activities were significantly decreased after 24 h . The decrease in cytochrome P450 activities was accompanied by losses of cytochrome P4501A and P4503A apoproteins . The microsomal glucuronidation rate of 1-naphthol was not affected in LPS-treated pigs . Comparing the LPS model with our previous findings in the Actinobacillus pleuropneumoniae model showed a remarkable similarity with regard to the effects on hepatic drug metabolism.

J Anim Sci, 1996 Oct, 74(10), 2431 - 40
Effects of immune challenge, dietary energy density, and source of energy on performance and immunity in weanling pigs; van Heugten E et al.; The objective of this study was to investigate the effects of nutrient density and dietary energy source on performance and immune function of weanling pigs that were either challenged or not challenged with Escherichia coli lipopolysaccharide (LPS) . A basal diet was formulated to contain 14 g CP/MJ DE and 7 g lysine/100 g CP . Sulfur amino acids, threonine and tryptophan were kept constant relative to lysine . Experimental diets were mixed using 70 parts basal diet and either 30 parts starch or an isocaloric amount (14 parts) of lard . Diets were fed either for ad libitum intake or on a pair-feeding basis to evaluate effects of diet nutrient density or source of energy, respectively . On d 9 and 25, pigs were challenged i.m . with either 1 mL of a LPS solution or a control solution . Lymphocyte blastogenesis was measured 2 d after the LPS administration and antibody response to sheep red blood cells (SRBC) or ovalbumin was determined 3 d after challenge . No interactive effects on performance were observed between LPS challenge and energy density or source of energy (P > .10) . Injection of LPS tended to reduce feed intake and daily gain (P < .10), but not efficiency of feed or energy utilization . Addition of fat to the diets improved feed efficiency and efficiency of energy utilization for gain (P < .05) . No consistent effects of LPS challenge, energy density, or source of energy were observed for lymphocyte blastogenesis . Antibody response to ovalbumin, but not to SRBC, was decreased by fat (P < .05) . Results indicate that increasing energy density of the diet did not alter the performance depression due to LPS challenge . Addition of fat to the diet improved feed efficiency and efficiency of energy conversion but may depress the humoral immune response . Effects of fat on the immune response may depend on the immune status of the pig.

Can J Vet Res, 1996 Oct, 60(4), 257 - 62
Polymerase chain reaction analysis of TNF-alpha and IL-6 mRNA levels in whole blood from cattle naturally or experimentally infected with Mycobacterium paratuberculosis; Adams JL et al.; Johne's disease is characterized by a chronic enteritis that results in granulomatous inflammation, cachexia, and eventual death of cattle infected with Mycobacterium paratuberculosis . The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes . The potential role of these cytokines in the development and progression of Johne's disease has not been investigated . Using the polymerase chain reaction (PCR) and specific bovine oligonucleotide cytokine primers and probes, we examined the expression of messenger RNA for these cytokines in whole blood from M . paratuberculosis infected and uninfected cattle . Cytokine mRNA levels were examined before and after in vitro incubation with E.coli lipopolysaccharide (LPS) and lipoarabinomannan (LAM) purified from M . paratuberculosis . Uninfected calves, experimentally infected calves, and naturally infected cattle all displayed similar cytokine mRNA expression patterns . However, individual animals demonstrated variability in the levels of IL-6 and TNF-alpha mRNA expression as determined by a semiquantitative PCR method using 32P-labelled oligonucleotide probes.

J Surg Res, 1996 Oct, 65(2), 149 - 58
Thrombin is a distal mediator of lipopolysaccharide-induced liver injury in the rat; Moulin F et al.; Previous results demonstrated that rats given Escherichia coli lipopolysaccharide (LPS; 4 mg/kg, i.v.) experience hepatocellular necrosis that begins within 4 hr and that prior treatment with anticoagulants (e.g., heparin) which target thrombin prevents the liver injury . In this study, hepatocellular injury, as marked by increased plasma alanine aminotransferase (ALT) activity and histologic changes, was prevented when heparin or hirudin was administered to rats shortly before the onset of injury . These results suggest that thrombin is a critical mediator that acts distally in the series of inflammatory events that culminates in hepatocellular damage . To explore further this hypothesis, livers isolated from rats 2 hr after LPS administration were perfused with various media . Perfusion of livers with medium comprising diluted blood from heparin-treated donors resulted in no release of ALT activity . By contrast, perfusion with similar medium anticoagulated with ancrod, which prevents clotting by depleting fibrinogen but does not inhibit thrombin, resulted in hepatocellular injury evidenced as a time-dependent appearance of ALT activity in the medium . Moreover, when livers from rats treated 2 hr previously with LPS were perfused with buffer to which thrombin had been added, injury resulted . No injury resulted when thrombin was omitted from the buffer or when livers from saline-treated rats were used . These results indicate that thrombin is a critical and distal mediator of LPS-induced liver damage and contributes to hepatocellular injury through a mechanism that is independent of clot formation . Furthermore, inflammatory events triggered by LPS exposure are a prerequisite for thrombin-induced injury.

J Surg Res, 1996 Oct, 65(2), 101 - 8
Transthoracic bioimpedance can measure extravascular lung water in acute lung injury; Nierman DM et al.; We used a porcine endotoxemic model of acute lung injury to compare extravascular lung water (EVLW) measured by right transthoracic bioimpedance to postmortem gravimetric EVLW measurements . Adult pigs were randomized into control (N = 5) or endotoxin groups {150 microgram/kg Escherichia coli lipopolysaccharide B for 1 hr followed by 3 hr of resuscitation for a thermodilution cardiac output less than 90% of baseline using either isotonic saline (N = 5) or isooncotic albumin (N = 5)} . Right lung resistance was measured using a novel electrode array and a highly sensitive analyzer and was used to calculate right lung resistivity . At the end of the experiment, animals in the endotoxin-albumin group had higher gravimetric EVLWs than those in the endotoxin-saline or control groups (P < 0.05) . Right lung resistivity corrected for body weight significantly correlated with gravimetric EVLW (r2 = 0.49; SEE = 0.96; P = 0.0038) . Using multiple regression analysis, a predictive equation for EVLW based on right lung resistivity, body weight, and mean pulmonary artery pressure was generated (r2 = 0.81; SEE = 0.60; P < 0.0001) . These results demonstrate that right lung resistivity measurements can provide a noninvasive estimate of EVLW . In addition, crystalloid may be preferable to colloid for fluid resuscitation in noncardiogenic pulmonary edema.

Shock, 1996 Oct, 6(4), 279 - 85
Effect of nitric oxide synthase inhibition on myocardial contractility in anesthetized normal and endotoxemic dogs; Kaszaki J et al.; Nitric oxide (NO) produced by the induced NO synthase (NOS) enzyme has been implicated in the mechanisms of the circulatory changes that occur in the later stages of sepsis . As NO produced by the constitutive form of the enzyme is known to play a role in the regulation of normal circulation, we have performed a series of experiments to study the early circulatory effects of inhibition of NOS in a hyperdynamic endotoxemic dog model . Pentobarbital-anesthetized animals were used . Cardiac output (CO) was measured by thermodilution . Myocardial contractility (MC) was estimated from the slope of the left ventricular end-systolic pressure-diameter relationship obtained from sonomicrometer- and catheter-tip manometer signals in closed chest animals . All animals received a 15 mL/kg/h infusion of Ringer's lactate . A hyperdynamic response was elicited by a 2 h infusion of a total dose of 5.3 micrograms/kg Escherichia coli O55:B5 endotoxin (ETX) . CO increased initially by about 25%, and total peripheral resistance decreased by 35% . These changes subsided in 60-90 min, after which a sustained decrease in CO occurred . MC elevated transiently by 25% after the first 30 min of ETX infusion, then decreased gradually below the control level . Administration of 2 mg/kg of the NOS inhibitor N-nitro-L-arginine (NNA) between the 45th and 55th min of the ETX infusion increased MC to the level in the control group, but accelerated the decline of the initially increased CO and caused a sustained increase in total peripheral resistance to about 50% above the control level . In normal (nonendotoxin treated) dogs, NNA also caused a similar increase in MC which, however, lasted at least 3 h . Left ventricular diameter increased in the NNA-treated groups . This increase also occurred in the endotoxin-only group but with a delay of about 2.5 h . Our results demonstrate the participation of constitutive NOS-produced NO in the early hyperdynamic response of endotoxemia . Suppression of NO is associated with increased myocardial contractility . NNA treatment may be favorable for the restoration of depressed cardiac contractility during endotoxemia, but this treatment is probably detrimental for the compensatory systemic flow (CO) increase.

Shock, 1996 Oct, 6(4), 267 - 73
Chronic endotoxemia and endothelium-dependent vasodilation in coronary arteries; Myers PR et al.; Endotoxin acutely decreases the production of nitric oxide, leading to abnormal regulation of coronary vascular tone: however, the effects of chronic endotoxemia on vasomotion are unknown . We therefore tested the hypothesis that chronic, low-level endotoxemia inhibits endothelium-dependent vasodilation . Rabbits were continuously infused with a subclinical dose of Escherichia coli endotoxin (.6 microgram/24 h, intraperitoneal) or saline for 5 wk . Endotoxin at this concentration elicited no significant sustained pyretic or hemodynamic responses . Both endothelium-dependent and independent vasomotor responses were determined in coronary arteries (250-500 mu) . Vasorelaxation in response to acetylcholine, but no adenosine diphosphate (ADP), was significantly enhanced in endotoxin-challenged animals (EC50 = 62.6 +/- 11.1 nM, control vs . 33.97 +/- 5.7 nM, endotoxin-challenged; p < .05) . Vasoconstriction to PGF2 alpha, but not KCl, was significantly decreased in endotoxin-challenged animals . These results indicate that endothelium-dependent and independent vasomotor responses are altered during chronic endotoxemia and are due, in part, to alterations in signal-transduction mechanisms specific for certain types of receptors.

Shock, 1996 Oct, 6(4), 254 - 8
Pteridine and nitrite/nitrate formation in experimental septic and traumatic shock; Strohmaier W et al.; Bacterial lipopolysaccharides (LPS) induce the activity of guanosine triphosphate (GTP)-cyclohydrolase I (GTP-CHI), the first enzyme in the biosynthesis of tetrahydrobiopterin (H4bip) from GTP in endothelial cells and macrophages . In these and other cells, LPS also acts costimulatory with cytokines, i.e., mainly tumor necrosis factor-alpha (TNF-alpha) . H4bip is the cofactor for nitric oxide synthase (NOS) . We were interested in comparing the pteridine and nitrate levels in two baboon models: a hyperdynamic sepsis model and a hemorrhagic traumatic shock model . Our results show a similar response of pteridines (H4bip, neopterin) and nitrite/nitrate levels to an immune stimulus . LPS, which peaks rapidly, induces a sustained increase in pteridine levels in septic animals . Since hemorrhagic animals show very little response in terms of cytokine production, it was not possible to measure the induction of neopterin and nitrite/nitrate . This information could aid our understanding of the regulatory mechanisms in various forms of experimental shock.

Chem Res Toxicol, 1996 Oct-Nov, 9(7), 1167 - 75
Comparison of the efficiency of synthesis past single bulky DNA adducts in vivo and in vitro by the polymerase III holoenzyme; Latham GJ et al.; Previous studies from our laboratory revealed that site-specific and stereospecific styrene oxide (SO) lesions in M13 DNA were readily bypassed when transfected into Escherichia coli cells, but these same lesions blocked the progress of several purified polymerases in vitro when situated in oligodeoxynucleotide templates (Latham, G . J., et al . (1993) J . Biol . Chem . 268, 23427-23434; Latham, G . J., et al . (1995) Chem . Res . Toxicol . 8, 422-430) . To resolve this apparent discrepancy, we constructed single-stranded M13 genomes containing single SO adducts and compared their replication efficiencies in E . coli cells to the extent of bypass synthesis in vitro using three different complexes of the purified E . coli polymerase III (Pol III) holoenzyme . The transformation efficiencies of the SO-adducted M13 templates were comparable to those of the nonadducted controls, indicating facile bypass in E . coli . When the identical adducted M13 vectors were replicated in vitro with the reconstituted complexes of the Pol III holoenzyme, the results were consistent with the in vivo data: Synthesis past two of the three SO adducts in M13 was unhindered relative to synthesis on the unadducted M13 control template . Since our previous in vitro assays indicated that SO adducts in 33-mer templates largely blocked polymerases other than Pol III, we repeated these studies using reconstituted Pol III . Significantly, Pol III replication was poorly processive and strongly terminated by SO lesions in 33-mer templates . This result was in stark contrast to the efficient bypass in vitro of the same adducts in M13 DNA . In fact, Pol III-mediated bypass was enhanced to > 75-fold on adducted circular M13 templates as compared to adducted linear oligodeoxynucleotides . The implications of the effects of polymerase processivity and template-primer structure upon lesion bypass are discussed.

Mol Microbiol, 1996 Oct, 22(1), 135 - 47
Involvement of the sensor kinase EnvZ in the in vivo activation of the response-regulator PhoB by acetyl phosphate; Kim SK et al.; Three signalling pathways lead to activation of the phosphate (Pho) regulon by phosphorylation of the response-regulator PhoB in Escherichia coli . One pathway responds to the extracellular inorganic phosphate (PI) level and leads to activation by the Pi sensor kinase, PhoR . The other two pathways are Pi independent and are apparent in the absence of PhoR . One Pi-independent pathway responds to the level of an unknown catabolite and leads to activation by the catabolite regulatory sensor kinase, CreC (originally called PhoM); the other Pi-independent pathway responds to acetyl phosphate and leads to activation by a process requiring acetyl phosphate . Here we show that activation of PhoB by acetyl phosphate can require the sensor kinase EnvZ . Accordingly, we propose that the in vivo activation of PhoB by acetyl phosphate (and perhaps other two-component response-regulators as well) probably always requires a certain kinase that can vary depending upon the growth conditions.

Mol Microbiol, 1996 Oct, 22(1), 53 - 61
Dual regulation of the paraquat-inducible gene pqi-5 by SoxS and RpoS in Escherichia coli; Koh YS et al.; The pqi-5 gene, producing a probable membrane protein of unknown function, has been reported to be a member of the soxRS regulon . The SoxRS-dependent induction of pqi-5 by paraquat occurs only during the exponential phase . The expression of pqi-5 increased in the absence of paraquat during the stationary phase or under conditions of carbon or phosphate starvation . This increase was regulated at the transcriptional level by RpoS (sigma S), which recognized the second promoter (P2) approx . 5 nucleotides upstream from the promoter (P1) used at the exponential phase . Studies with a series of 5' deletions revealed that the paraquat-responsive element resides between -52 and -42 nucleotides upstream from the P1 start site, whose nucleotide sequence matches closely to other SoxS-binding sequences . The stationary-phase induction required sequences up to position -42, which correspond to the 5' border of the putative -35 hexamer for the P2 promoter . The binding of the purified SoxS protein to the pqi-5 promoter upstream sequences was demonstrated by gel mobility-shift and DNase I protection assays . The transcription from P1 promoter by E sigma D was activated by purified SoxS in vitro, as was observed in vivo . The dual regulation of pqi-5 by SoxS at the exponential phase and RpoS at the stationary phase is the first to be reported among the members of the soxRS regulon, suggesting that this gene might indeed play some role under stressful conditions.

Mol Microbiol, 1996 Oct, 22(1), 21 - 9
FIS is a regulator of metabolism in Escherichia coli; Gonzalez-Gil G et al.; The Escherichia coli DNA-binding protein FIS (factor for inversion stimulation) stimulates site-specific recombination reactions catalysed by DNA invertases and is an activator of stable RNA synthesis . To address the question of whether FIS is involved in other cellular processes we have identified and sequenced proteins whose expression pattern is affected by FIS . This has led to the identification of several E . coli genes whose expression in vivo is either enhanced or repressed by FIS . All of these genes encode enzymes or transport proteins involved in the catabolism of sugars or nucleic acids, and their expression is also dependent on the cAMP-CRP complex . In most cases studied the regulation by FIS is indirect and occurs through effects on the synthesis of the respective repressor proteins . We conclude that FIS is a transcriptional modulator involved in the regulation of metabolism in E . coli.

Eur J Immunol, 1996 Oct, 26(10), 2440 - 7
Biological properties of recombinant chicken interferon-gamma; Weining KC et al.; Supernatants of the chicken T cell line 855 contain antiviral and macrophage activating factor activity and strongly activate transcription of the guanylate binding protein (GBP) gene in chicken cells . To characterize the cytokine responsible for the GBP-inducing activity, we chose a cDNA expression cloning strategy in COS cells . Sequencing a positive clone revealed that it encode chicken interferon-gamma (ChIFN-gamma) . Histidine-tagged ChIFN-gamma was expressed in Escherichia coli and purified by nickel chelate affinity chromatography . ChIFN-gamma from COS cells and E . coli both potently induced GBP RNA synthesis but were rather poor antiviral agents . In macrophages, recombinant ChIFN-strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen . A rabbit antiserum to E . coli derived ChIFN-gamma effectively neutralized the macrophage-activating factor activity secreted by concanavalin A-induced spleen cells and various T cell lines, suggesting that IFN-gamma is the major macrophage-activating factor of the chicken.

Eur J Biochem, 1996 Oct 1, 241(1), 272 - 9
Cloning, expression and properties of the alpha' subunit of casein kinase 2 from zebrafish (Danio rerio); Antonelli M et al.; The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division . The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2) . The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter . There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F . J . & Pyerin, W . (1994) Recombinant human casein kinase II . A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur . J . Biochem . 220, 263-273) . In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported . The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish . The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis . The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1) . The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide . However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha . The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity . The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition . Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit . When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D . rerio) and alpha (X . laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X . laevis) only 7-8-fold.

Eur J Biochem, 1996 Oct 1, 241(1), 201 - 7
Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase; Lubkowski J et al.; The amino acid sequence and tertiary structure of Wolinella succinogenes L-asparaginase were determined, and were compared with the structures of other type-II bacterial L-asparaginases . Each chain of this homotetrameric enzyme consists of 330 residues . The amino acid sequence is 40-50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical . Differences between the amino acid sequence of W . succinogenes L-asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity . The overall fold of the protein subunit is almost identical to that observed for other L-asparaginases . Two fragments in each subunit, a very highly flexible loop (approximately 20 amino acids) that forms part of the active site, and the N-terminus (two amino acids), are not defined in the structure . The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active-site pocket . The rigid part of the active site, which includes the asparaginase triad Thr93-Lys 166-Asp94, is structurally very highly conserved with equivalent regions found in other type-II bacterial L-asparaginases.

Eur J Biochem, 1996 Oct 1, 241(1), 193 - 200
Protein phosphatase activity of abscisic acid insensitive 1 (ABI1) protein from Arabidopsis thaliana; Bertauche N et al.; Mutations at the ABI1 (abscisic acid insensitive 1) locus of the plant Arabidopsis thaliana cause a reduction in sensitivity to the plant hormone abscisic acid . The sequence of ABI1 predicts a protein composed of an N-terminal domain that contains motifs for an EF-hand Ca(2+)-binding site, and a C-terminal domain with similarities to protein serine/threonine phosphatases 2C . We report here two sets of experimental evidence that indicate that ABI1 has typical protein phosphatase 2C activity . First, expression of the ABI1 C-terminal domain partially complemented the temperature-sensitive growth defect of a Saccharomyces cerevisiae protein phosphatase 2C mutant . Second, recombinant proteins that contained the ABI1 C-terminal domain displayed in vitro phosphatase activity towards 32P-labelled casein, and this activity displayed Mg2+ or Mn2+ dependence and okadaic acid insensitivity typical of protein phosphatases 2C . Characterisation of recombinant proteins that contained various portions of ABI1 indicated that the putative EF-hand motif is unlikely to mediate Ca2+ regulation of the ABI1 phosphatase activity at physiological Ca2+ concentrations, and may represent in EF-hand analogue rather than an EF-hand homologue . The abil-l mutation appeared to cause significant reduction in the phosphatase activity of ABI1 . These results are discussed in relation to the dominant phenotype of abil-l over the wild-type allele in plants, and to the possible role of ABI1 in abscisic acid signalling.

Eur J Biochem, 1996 Oct 1, 241(1), 162 - 70
Properties of the purified, recombinant, NADP(H)-binding domain III of the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum; Diggle C et al.; Transhydrogenase comprises three domains . Domains I and III are peripheral to the membrane and possess the NAD(H)- and NADP(H)-binding sites, respectively, and domain II spans the membrane . Domain III of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified . The purified protein was associated with substoichiometric quantities of tightly bound NADP+ and NADPH . Fluorescence spectra of the domain III protein revealed emissions due to Tyr residues . Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close . The rate constants for NADP+ release and NADPH release from domain III were 0.03 s-1 and 5.6 x 10(4) s-1, respectively . In the absence of domain II a mixture of the recombinant domain III protein, plus the previously described recombinant domain I protein, catalysed reduction of acetylpyridine-adenine dinucleotide (AcPdAD+) by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP+ from domain III . Similarly, the mixture catalysed reduction of thio-NADP+ by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain III . The mixture also catalysed very rapid reduction of AcPdAD+ by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H) . Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain III and vice versa indicated (a) that during reduction of AcPdAD+ by NADPH, a single domain I protein can visit and transfer H equivalents to about 60 domain III proteins during the time taken for a single domain III to release its NADP+, whereas (b) the cyclic reaction is rapid on the timescale of formation and break-down of the domain I . III complex . The rate of the hydride transfer reaction was similar in the domain I.III complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I.III complex due to the greatly decreased rates of release of NADP+ and NADPH . It is concluded that, in the complete enzyme, conformational changes in the membrane-spanning domain II, which result from proton translocation, lead to changes in the binding affinity of domain III for NADP+ and for NADPH.

Eur J Biochem, 1996 Oct 1, 241(1), 149 - 54
The corrinoid-containing 23-kDa subunit MtrA of the energy-conserving N5-methyltetrahydromethanopterin:coenzyme M methyltransferase complex from Methanobacterium thermoautotrophicum . EPR spectroscopic evidence for a histidine residue as a cobalt ligand of the cobamide; Harms U et al.; N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum is a membrane-associated enzyme complex that catalyzes an energy-conserving, sodium ion translocating step in methanogenesis from H2 and CO2 . The complex is composed of eight different subunits, MtrA-H, one of which (MtrA) harbours a corrinoid as prosthetic group . In this study, we report the structural properties of MtrA1 {des-(214-239)-MtrA}, which is a deletion mutant of MtrA that lacks the last 25 C-terminal hydrophobic amino acids rendering the membrane protein soluble: (a) mtrA1 was heterologously expressed in Escherichia coli . Overexpression yielded a cytoplasmic protein which was purified approximately tenfold to apparent homogeneity . The purified protein was devoid of its corrinoid prosthetic group and not correctly folded as was evident from its electrophoretic mobility in SDS/PAGE . (b) Unfolding of MtrA1 with guanidine/HCl and refolding in the presence of cobalamin resulted in the formation of the correctly folded MtrA1 holoprotein that contained tightly bound cob(II)-alamin; the rate of reconstitution was highest when the refolding proceeded in the presence of titanium(III) citrate, which suggested that cob(I)alamin is the corrinoid species that binds to the apoprotein . (c) EPR spectra of the cob(II)alamin-containing holoprotein differentially labelled with 14N (nuclear spin 1) and 15N (nuclear spin 1/2) revealed that the corrinoid is bound to MtrA1 in the base-off form and that the Co(II) of the prosthetic group is coordinated by a histidine residue of the apoprotein . The results are interpreted with respect to the mechanism of energy conservation by the MtrA-H complex.

Eur J Biochem, 1996 Oct 1, 241(1), 133 - 41
Affinity labeling of Escherichia coli histidyl-tRNA synthetase with reactive ATP analogues . Identification of labeled amino acid residues by matrix assisted laser desorption-ionization mass spectrometry; Gillet S et al.; Recent affinity labeling studies have revealed that dimeric histidyl-tRNA synthetase from Escherichia coli displayed half-of-the-sites reactivity toward labeling with pyridoxal 5'-phosphate {Kalogerakos, T., Hountondji, C., Berne, P . F., Dutka, S . & Blanquet, S . (1994) Biochimie (Paris) 76, 33-44} . In the present report, affinity labeling studies were conducted by using other ATP analogues such as pyridoxal 5'-diphospho-5'-adenosine (pyridoxal-ppAdo), pyridoxal 5'-triphospho-5'-adenosine (pyridoxal-pppAdo), pyridoxal 5'-diphosphate (pyridoxal-P2) and 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo) . The histidine-dependent isotopic {32P}PP/ATP exchange activity of His-tRNA synthetase was rapidly and completely lost upon incubation with either pyridoxal-ppAdo, pyridoxal-pppAdo or pyridoxal-P2, followed by reduction with sodium borohydride . Complete inactivation of His-tRNA synthetase corresponded to the incorporation of 2.8 mol of either pyridoxal-ppAdo or pyridoxal-P2/mol dimeric synthetase . Incubation of His-tRNA synthetase with FSO2BzAdo also resulted in a complete inactivation of the synthetase . However, contrasting with the pyridoxal derivatives, the plot of the residual enzymatic activity against the amount of covalently bound FSO2BzAdo appeared biphasic . In the early stages of inactivation, the relationship between the amount of residual activity and FSO2BzAdo incorporation was linear and extrapolated to a stoichiometry of 1.1 mol reagent/mol His-tRNA synthetase, suggesting that the labeling of one subunit was sufficient to inactivate one dimeric His-tRNA synthetase molecule . At longer incubation periods, additional reagent incorporation occurred and culminated at 2.5 mol label/mol His-tRNA synthetase . Excess of MgATP protected the enzyme against inactivation by either studied reagent . The labeled amino acid residues were identified by matrix-assisted-laser-desorption-ionization mass spectrometry, by measuring the peptide mass increase caused by the reagents . An identical set of four lysyl residues (Lys2, Lys118, Lys369 and Lys370 of His-tRNA synthetase) was found attached to pyridoxal-ppAdo or pyridoxal-P2 . In addition, pyridoxal-ppAdo labeled the alpha-amino group of the N-terminal alanine . In a His-tRNA synthetase sample having incorporated 2.5 mol FSO2BzAdo/mol), the labeled amino acid residues were Lys118, Lys196, Tyr262 (or Tyr263), Lys369 and Lys377 . Whatever the used reagent, Lys118 appeared to be the predominantly labeled residue, Lys118 belongs to fragment 112-124 (RHERPQK-GRYRQF) corresponding to motif 2 of class 2 aminoacyl-tRNA synthetases . The other modified lysyl residues (lysines 369, 370 and 377) are close to the catalytic motif 3, in the C-terminal region of the synthetase . Tyr262 and Tyr263 belong to a fragment 256-263 (LVRGLDYY) highly conserved among all known His-tRNA synthetase primary structures . Examination of the recently solved structure of crystalline E . coli His-tRNA synthetase {Amez, J . G., Harris, D . C., Mitschler, A., Rees, B., Francklyn, C . S . & Moras, D . (1995) EMBO J . 14, 4143-4155} shows that, with the exception of lysines 369, 370 and 377, the location of which may account for peculiar accessibility and reactivity, all the amino acid residues identified in this study map near the enzyme nucleotide-binding site, at the N-terminal catalytic domain of the synthetase.

Eur J Biochem, 1996 Oct 1, 241(1), 64 - 9
Ferritin mRNAs in Schistosoma mansoni do not have iron-responsive elements for post-transcriptional regulation; Schussler P et al.; Schistosoma mansoni possesses two isoforms of ferritin, soma and yolk ferritin . The soma ferritin occurs at a low level in most cells of both genders, whereas the yolk ferritin is a female-specific gene product that is expressed at high level in the vitellarium . In higher animals, ferritin mRNA is regulated by iron via the interaction of cytoplasmic binding proteins (IRPs) with a specific sequence element in the 5' untranslated region (UTR) referred to as the iron-responsive element (IRE) . Sequence studies of the 5' UTRs, gel retardation assays, and hybridization experiments show that neither ferritin mRNAs of S . mansoni is regulated by an IRE/IRP mechanism . It is suggested that ferritins in schistosomes are controlled only at the transcriptional level.

Eur J Biochem, 1996 Oct 1, 241(1), 6 - 11
Lipid body lipoxygenase characterized by protein fragmentation, cDNA sequence and very early expression of the enzyme during germination of cucumber seeds; Hohne M et al.; Lipid bodies are cellular compartments containing triacylglycerols . They are encompassed by a phospholipid monolayer and decorated with characteristic proteins . In plants, lipid bodies are synthesized during seed formation but acquire new proteins during seed germination . In germinating cucumber (Cucumis sativus) seeds, the set of newly synthesized proteins appearing in the lipid bodies at the early stage of triacylglycerol mobilization comprises a special form of lipoxygenase . We isolated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogen bromide . A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lipoxygenase forms during germination . This allowed a clear distinction of this enzyme from other lipoxygenase isoforms . Hence, for determining the molecular structure of lipid body lipoxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day 1 of germination . From the cDNA sequence, oligonucleotides were derived that specifically detected lipid body lipoxygenase mRNA on northern blots . The very early expression of lipid body lipoxygenase was corroborated by this approach . Good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically . In particular, the cleavage products of cyanogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA . The sequence data show a lipoxygenase form characterized by a molecular mass of 99655 Da, which is significantly higher than the molecular masses of the cytosolic forms . Compared to the cytosolic forms that exhibit a molecular mass of 95 kDa, the lipid body form has an N-terminal extension of 34 amino acid residues . No evidence for a cotranslational or post-translational proteolytic processing was obtained by the size comparison of the in vitro-translated lipoxygenase and the lipid body form.

Am J Physiol, 1996 Oct, 271(4 Pt 2), H1296 - 301
L-arginine augments nitric oxide production and mesenteric blood flow in ovine endotoxemia; Allman KG et al.; We studied the effects of administrating the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), or the nitric oxide precursor, L-arginine, on hemodynamic variables and serum nitrate concentrations in an anesthetized ovine model of endotoxemia to assess the effects on regional visceral blood flow and to determine whether L-arginine availability limits nitric oxide production . Animals received Escherichia coli endotoxin (2 micrograms/kg) followed 2 h later by L-NAME (25 mg/kg), L-arginine (0.575 g/kg), or saline administered over 1 h followed by an infusion of the same dose over 8 h (n = 6 per group) . Renal and mesenteric blood flow were measured by placement of electromagnetic flow probes, and serum nitrate concentrations were determined using vanadium III chloride or nitrate reductase reduction to nitric oxide or nitrite, respectively . The results showed L-NAME significantly increased systemic vascular resistance (P < 0.01), decreased serum nitrate concentrations (P < 0.05), and caused a transient reduction in mesenteric blood flow (P < 0.05) . L-Arginine caused a reduction in systemic vascular resistance (P < 0.01), increased mesenteric blood flow (P < 0.001) and conductance (P < 0.05) . There were no significant changes in renal arterial blood flow in either group . We conclude that the availability of L-arginine limits nitric oxide production in endotoxemia and, furthermore, that L-arginine administration in this model causes significant mesenteric vasodilatation . L-NAME administration had only limited effect on visceral blood flow despite a marked increase in systemic vascular resistance and a reduction in nitric oxide production.

Am J Physiol, 1996 Oct, 271(4 Pt 1), G539 - 48
Inhibition of nitric oxide synthase gene expression in vivo and in vitro by repeated doses of endotoxin; Chang CC et al.; We have examined the effects of repeated endotoxin administration in vivo and in vitro on the induction of nitric oxide synthase (NOS) . In vivo, hepatic NOS activity and mRNA were increased markedly by the administration of Escherichia coli lipopolysaccharide (LPS) . The change in hepatic NOS activity coincided with a marked accumulation of hepatic citrulline . Both enzyme activity and citrulline concentration returned to normal by 12 h after LPS administration . At this time, a subsequent administration of endotoxin caused no change in either NOS mRNA, NOS activity, or citrulline concentration, and thus an endotoxin-refractory state for nitric oxide (NO) synthesis was established . Normal sensitivity was reestablished by 24 h after the initial dose . In vitro studies using both a macrophage cell line (HD11) and primary macrophages indicated that LPS pretreatment caused cells in culture to become completely refractory to subsequent stimulation by LPS . Finally, we tested the hypothesis that NO may be involved in the development of the refractory state . Various inhibitors blocked the initial synthesis of NO by > 90% but failed to influence the development of the refractory state . Our study demonstrates both in vivo and in vitro that NO synthesis is completely blocked after repeated exposure to endotoxin by a mechanism that appears to be pretranslational . This model of early endotoxin tolerance may provide insight into the molecular mechanisms that regulate expression of the NOS gene.

Protein Sci, 1996 Oct, 5(10), 2125 - 7
Crystallization and preliminary X-ray analysis of the monomeric Cu,Zn superoxide dismutase from Escherichia coli; Battistoni A et al.; The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E . coli strain BMH 71/18 . The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs . Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2(1), with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, beta = 111.4 degrees . One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.

Protein Sci, 1996 Oct, 5(10), 2089 - 94
In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state; Subramaniam V et al.; When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered . In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms) . The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein . In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h . Our results are in agreement with the report by Hattori et al . (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.

Protein Sci, 1996 Oct, 5(10), 1984 - 90
The 1.8-A X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding; Sugiyama S et al.; The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm . This component specifically binds either spermidine or putrescine . The crystal structure of the E . coli PotD protein complexed with spermidine was solved at 1.8 A resolution and revealed the detailed substrate-binding mechanism . The structure provided the detailed conformation of the bound spermidine . Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain . Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211 . Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine . To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure . The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine . On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.

Protein Sci, 1996 Oct, 5(10), 1973 - 83
Protein secondary structural types are differentially coded on messenger RNA; Thanaraj TA et al.; Tricodon regions on messenger RNAs corresponding to a set of proteins from Escherichia coli were scrutinized for their translation speed . The fractional frequency values of the individual codons as they occur in mRNAs of highly expressed genes from Escherichia coli were taken as an indicative measure of the translation speed . The tricodons were classified by the sum of the frequency values of the constituent codons . Examination of the conformation of the encoded amino acid residues in the corresponding protein tertiary structures revealed a correlation between codon usage in mRNA and topological features of the encoded proteins . Alpha helices on proteins tend to be preferentially coded by translationally fast mRNA regions while the slow segments often code for beta strands and coil regions . Fast regions correspondingly avoid coding for beta strands and coil regions while the slow regions similarly move away from encoding alpha helices . Structural and mechanistic aspects of the ribosome peptide channel support the relevance of sequence fragment translation and subsequent conformation . A discussion is presented relating the observation to the reported kinetic data on the formation and stabilization of protein secondary structural types during protein folding . The observed absence of such strong positive selection for codons in non-highly expressed genes is compatible with existing theories that mutation pressure may well dominate codon selection in non-highly expressed genes.

Graefes Arch Clin Exp Ophthalmol, 1996 Oct, 234(10), 643 - 7
Hemoglobin exacerbates the ocular inflammatory response to endotoxin; McGahan MC et al.; BACKGROUND: There is a clinical impression that bleeding into sites of inflammation exacerbates the inflammatory response . It has been hypothesized that hemoglobinic iron (Fe) contributes to this response by catalyzing free radical reactions . In the present study, the effects of autologous hemoglobin on the inflammatory response to endotoxin was determined . In addition, the possible contributions of Fe to this response was assessed by co-injection of either transferrin or desferrioxamine . METHODS: A mild ocular inflammation was induced in rabbits by intravitreal injection of 0.25 ng endotoxin . In some animals apotransferrin, hemoglobin, hemoglobin + apotransferrin or hemoglobin + desferrioxamine were co-injected . Twenty-four hours later, anterior uveitis was quantified by slit-lamp examination and determination of protein concentration and infiltration of white cells into the aqueous humor . RESULTS: Co-injection of autologous hemoglobin with endotoxin greatly exacerbated the ocular inflammatory response to endotoxin, especially the infiltration of white cells, which was increased 15-fold . Both apotransferrin, which binds Fe at high affinity, and desferrioxamine, which chelates Fe, greatly decreased the cellular response to the co-injection . CONCLUSIONS: It is likely that hemoglobinic Fe is responsible for the increased infiltration of white cells caused by the co-injection of autologous hemaglobin and endotoxin.

EMBO J, 1996 Oct 1, 15(19), 5449 - 58
Influence of DNA geometry on transcriptional activation in Escherichia coli; Dethiollaz S et al.; Transcription from many Escherichia coli promoters can be activated by the cAMP-CRP complex bound at different locations upstream of the promoter . At some locations the mechanism of activation involves direct protein-protein contacts between CRP and the RNA polymerase . We positioned the CRP binding site at various distances from the transcription start site of the malT promoter and measured the in vivo activities of these promoter variants . From the activation profiles we deduce that the protein-protein interactions involved in transcriptional activation are rather rigid . A heterologous protein (IHF) that bends the DNA to a similar degree as does CRP activates transcription when bound at sites equivalent to activating positions for CRP . DNA geometry makes a major contribution to the process of transcriptional activation and DNA upstream of the activator binding site participates in this process . Removal of this DNA decreases the capacity of the malT promoter to be activated by CRP in vitro . We conclude that both DNA topology and direct protein-protein contacts contribute to transcriptional activation and that the relative importance of these two modes of activation depends on the nature of the activator and on the location of the activator binding site.

EMBO J, 1996 Oct 1, 15(19), 5209 - 17
Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase; Prinz WA et al.; Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus . Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence . In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant . The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase . These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence . These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm . We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export . These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.

Carcinogenesis, 1996 Oct, 17(10), 2249 - 52
Release of sulfur mustard-modified DNA bases by Escherichia coli 3-methyladenine DNA glycosylase II; Matijasevic Z et al.; The toxic effects of sulfur mustard have been attributed to DNA modification with the formation of 7-hydroxyethylthioethyl guanine, 3-hydroxyethylthioethyl adenine and the cross-link, di-(2-guanin-7-yl-ethyl)sulfide . To investigate the action of bacterial 3-methyladenine DNA glycosylase II (Gly II) on these adducts, calf thymus DNA was modified with {14C}sulfur mustard and used as a substrate for Gly II . Gly II releases both 3-hydroxyethylthioethyl adenine and 7-hydroxyethylthioethyl guanine from this substrate . In comparison with the activity of Gly II towards methylated DNA, 3-hydroxyethylthioethyl adenine is released somewhat more slowly than 3-methyladenine, while 7-hydroxyethylthioethyl guanine is released much more readily than 7-methylguanine . Glycosylase action may play a role in protecting cells from the toxic effects of sulfur mustard.

Carcinogenesis, 1996 Oct, 17(10), 2177 - 82
Distinct substrate preference of human and mouse N-methylpurine-DNA glycosylases; Roy R et al.; N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair protein, removes several N-alkylpurine adducts, hypoxanthine, cyclic ethenoadducts of adenine, guanine and cytosine and 8-oxoguanine from DNA . The recombinant human and mouse MPGs, purified from Escherichia coli, show a significant difference in substrate preference . While both proteins prefer 3-methyladenine over other N-alkylpurines in DNA, the mouse MPG removes 7-methylguanine and 3-methylguanine at an approximately 2- to 3-fold higher rate than the human protein when adjusted for equal activity for the release of 3-methyladenine from DNA . Hybrid recombinant proteins containing N-terminal and C-terminal halves of the human and mouse glycosylases were partially purified from MPG-negative E.coli . Their substrate preferences suggest that the N-terminal half is more critical for the recognition of 3-methylguanine and 7-methylguanine.

Inflammation, 1996 Oct, 20(5), 555 - 68
Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides; Noguchi K et al.; The purpose of the present study was to investigate the involvement of cyclooxygenase-1(COX-1) and cyclooxygenase-2 (COX-2) in PGE2 production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria . LPS were isolated from Porphyromonas gingivalis (P . gingivalis), Actinobacillus actinomycetemcomitans (A . actinomycetemcomitans) and Eschericia coli (E coli) by the phenol-water procedure . The three LPS preparations produced PCE2 up to 48 h in a time-dependent manner in human gingival fibroblasts . P . gingivalis-LPS was the most potent stimulator of PGE2 production and, to a lesser extent, A actinomycetemcomitans- and E coli-LPS . Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE2 production . Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE2 production . Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h after P gingivalis-LPS stimulation, while expression of COX-1 protein was not affected by P . gingivalis-LPS . In order to investigate the regulation of PGE2 production . P . gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases . Both the inhibitors significantly inhibited PGE2 production . Herbimycin A treatment depressed expression of COX-2 protein . These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE2 not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE2 may be involved in the pathogenesis of periodontal diseases.

Hum Gene Ther, 1996 Oct 1, 7(15), 1883 - 93
Regulatable promoters for use in gene therapy applications: modification of the 5'-flanking region of the CFTR gene with multiple cAMP response elements to support basal, low-level gene expression that can be upregulated by exogenous agents that raise intracellular levels of cAMP; Suzuki M et al.; This study focuses on the design, construction, and evaluation of a chimeric promoter for gene therapy applications where it is desirable to have low-level basal expression of the newly transferred gene, which can be induced to higher levels of expression by the administration of pharmacologic agents that can be safely used locally and/or systemically in humans . To achieve this, a chimeric promoter was constructed using fragments of the 5'-flanking region of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, and multiple tandem repeats of the consensus sequence and flanking elements of the cAMP response element (CRE), promoter sequences that support increased transcription in response to elevations in intracellular cAMP levels . Preliminary studies using plasmid vectors demonstrated that: (i) the 5'-flanking sequences from the CFTR gene have low promoter activity in the human airway epithelial cell lines; (ii) chimeras using -718 bp fragment from the 5'-flanking sequence of CFTR gene as the base, with the addition of 4-10 units of a 25-bp sequence containing the CRE consensus sequence, were all inducible by a rise in intracellular cAMP, with the chimera having eight CRE repeats the most responsive; and (iii) a CF126(CRE8) chjimera, consisting of the -126 bp fragment from the 5'-flanking region of CFTR gene together with eight CRE repeats, yielded low-level basal activity but maximal upregulation by cAMP, resulting in expression of the reporter gene that was 51-58% of an RSV-LTR control . On the basis of these observations, replication-deficient adenoviral vectors containing the CF126(CRE8) chimera and the luciferase reporter gene {AdCF126(CRE8).Luc} or the Escherichia coli lacZ (beta-Gal) reporter gene {AdCF126(CRE8) . beta gal} were constructed . In several human airway epithelial cell lines, the AdCF126 (CRE) . Luc vector provided low basal activity, but was significantly upregulated by agents that increase cAMP levels . Intranasal administration of the beta-Gal-expressing AdCF126(CRE8) beta gal vector into C57B1/6 mice demonstrated cAMP-induced upregulation of the reporter gene in airway epithelial cells . Quantification of the inducibility of the basal promoter activity in the airway epithelium using the AdCF126(CRE8) . Luc vector demonstrated an 11-fold upregulation of the basal promoter activity in the lung with the administration of a phosphodiesterase inhibitor and a cAMP analog . These observations demonstrate the feasibility of using a chimeric promoter comprised of a minimal fragment of the CFTR 5'-flanking region, together with added multiple CRE, to control genes delivered in vivo . Importantly, because there are many drugs used in humans that raise cAMP, the concept of using a cAMP-regulatable promoter may also be a useful approach to enhance the safety, efficacy, and feasibility of a variety of human gene therapy strategies.

Hum Gene Ther, 1996 Oct 1, 7(15), 1853 - 60
Cancer gene therapy using tumor cells infected with recombinant vaccinia virus expressing GM-CSF; Qin H et al.; The efficacy of a recombinant vaccinia virus (rvv-mGM-CSF) expressing murine granulocyte-macrophage colony stimulating factor (GM-CSF) for use in cancer gene therapy was evaluated . C57BL/6 mice with established B16-F10 melanoma were treated by s.c . injection of irradiated B16 cells infected with two different recombinant vaccinia virus (rvv) constructs . Mice treated with rvv-mGM-CSF vaccine survived longer (p < 0.05), were free of palpable tumors (> 4 mm) longer (p < 0.02), and had smaller mean tumor volumes (p < 0.005) compared to those treated with irradiated B16 cells infected with a control rvv (rvv-lacZ) expressing Escherichia coli beta-galactosidase or irradiated uninfected B16 cells . The vaccine appeared to be B16 tumor cell specific, because there was no therapeutic effect when heterologous but syngeneic (H-2b) colon adenocarcinoma cells, MC-38 infected with rvv-mGM-CSF were used as vaccine . In this model, rvv expressing interleukin-2 (IL-2) was ineffective . In addition, experimental lung metastasis of B16 tumor cells was significantly inhibited by rvv-mGM-CSF vaccine compared to several control vaccines when the vaccine was applied either by i.p . route (p < 0.006) or by s.c . injection (p < 0.0008) . B16 cells expressing mGM-CSF after infection with rvv-mGM-CSF or transduction with a retroviral vector, were equally effective (p > 0.14) as vaccines against lung metastasis . Inhibition of metastasis was also B16 tumor cell specific . These data suggest that this approach of cancer gene therapy has a potential for use in cancer patients.

Br J Pharmacol, 1996 Oct, 119(3), 479 - 86
Effect of selective blockade of endothelin ETB receptors on the liver dysfunction and injury caused by endotoxaemia in the rat; Ruetten H et al.; 1 . We investigated the effects of the selective endothelin (ET)A receptor antagonist BQ-485 and the selective ETB receptor antagonist BQ-788 on circulatory failure, multiple organ dysfunction syndrome (MODS) and the alterations in acid base balance caused by endotoxaemia in the anaesthetized rat . 2 . Male Wistar rats were anaesthetized (thiopentone sodium; 120 mg kg-1, i.p.) and received a continuous infusion of vehicle (saline, 0.6 ml kg-1h-1, i.v.), BQ-485 (10 nmol kg-1 min-1, i.v.) or BQ-788 (10 nmol kg-1 min-1, i.v.) . Fifteen min later, animals received a bolus injection of either saline (0.9% NaCl, 1 ml kg-1, i.v.) or E . coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.) . 3 . Injection of LPS resulted in a fall in blood pressure from 115 +/- 4 mmHg (time 0) to 82 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the pressor responses to noradrenaline (NA, 1 microgram kg-1, i.v.) . Infusion of BQ-788 attenuated the delayed hypotension (at 360 min: 100 +/- 4 mmHg, n = 7; P < 0.05) and significantly enhanced the pressor responses elicited by NA (at 60 to 240 min) . In contrast, treatment of LPS-rats with BQ-485 augmented the hypotension (at 360 min), but did not affect the vascular hyporeactivity elicited by endotoxaemia . 4 . Endotoxaemia for 360 min resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), glutamate-oxalate-transferase (GOT) and glutamate-pyruvate-transferase (GPT) (indicators of hepatocellular injury), and bilirubin and gamma-glutamyl transferase (gamma GT) (indicators of liver failure) as well as nitrite (indicator of the induction of nitric oxide synthase; iNOS) . Treatment of LPS-rats with BQ-788, but not with BQ-485, attenuated the degree of liver injury and failure, while neither BQ-788 nor BQ-485 affected the acute renal failure or the induction of iNOS caused by endotoxin . 5 . Endotoxaemia also caused (within 15 min) an acute metabolic acidosis (falls in pH, HCO3-and base excess) which was compensated by hyperventilation (fall in PaCO2) . Treatment of LPS-rats with BQ-788 or BQ-485 did not affect the metabolic acidosis caused by LPS . 6 . Thus, the selective ETB receptor antagonist BQ-788 attenuated (i) the delayed hypotension, (ii) the vascular hyporeactivity to NA as well as (iii) the degree of hepatocellular injury and dysfunction caused by endotoxin in the anaesthetized rat . In contrast, the selective ETA receptor antagonist did neither attenuate the circulatory failure nor the liver or renal dysfunction associated with endotoxaemia . We propose that the prevention of the hepatocellular dysfunction and injury caused BQ-788 in endotoxaemia is due to an improvement in oxygen delivery to the liver secondary to (i) inhibition of pre-sinusoidal constriction, (ii) inhibition of sinusoidal constriction, and (iii) improvement in perfusion pressure.

Mol Biochem Parasitol, 1996 Oct 1, 80(2), 193 - 202
A chromatin-associated protein is encoded in a genomic region highly conserved in the Plasmodium genus; Birago C et al.; A single copy gene, pbB7, encoding a putative 26 kDa acidic protein has been isolated from Plasmodium berghei and appears to be part of a genomic region well conserved within the Plasmodium genus . The deduced amino acid sequence exhibits significant blocks of similarity with nucleosome assembly proteins from yeast and man . The nuclear localization of the natural protein and its close association with chromatin during the entire erythrocytic cycle of the parasite have been demonstrated using specific monoclonal antibodies against the pbB7 product expressed in Escherichia coli . These results suggest an involvement of this nuclear factor in the dynamics of chromatin packaging.

J Cell Biochem, 1996 Oct, 63(1), 61 - 73
Recombinant PSP94 (prostate secretory protein of 94 amino acids) demonstrates similar linear epitope structure as natural PSP94 protein; Xuan JW et al.; PSP94 has the potential to be a useful diagnostic marker and therapeutic agent in prostate cancer . Recently, different immunoassay systems for quantitative analysis of PSP94 in clinical samples have been developed, but the epitope structure of PSP94 protein has not been elucidated . In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein . GST-PSP94 contains antigenic determinants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the structure of natural PSP94 antigen . Since GST-PSP94 was expressed in E . coli and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the primary amino acid sequence, rather than conformational structure . This hypothesis was supported by reciprocal competition in ELISA among natural, GST-PSP94 fusion protein, and purified recombinant PSP94 protein . The results demonstrate that the various forms of PSP94 can compete with each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity . When natural and recombinant PSP94 protein were denatured in vitro with urea and alkali, no effect on the binding to antibody was found . The epitope activity of natural PSP94 was also shown to be resistant to the treatment of detergent and reducing agent . The location of one of the linear epitopes recognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequences . Competitive ELISA between the N-terminal peptide and PSP94 protein indicate that both natural and GST-PSP94 have similar immunoactive N-termini.

Cell Growth Differ, 1996 Oct, 7(10), 1415 - 24
Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences; Steel LF et al.; Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF . Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs) . To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region . Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR . We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs . Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect . The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides . The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation . Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Biotechniques . 1996 Oct;21(4):664, 666, 668, 670, 672.
Fluorometric assay for DNA polymerases and reverse transcriptase; Seville M et al.; We report a quick, easy and inexpensive fluorometric assay that measures the activity of replication enzymes using Pico-GreenTM . The systems tested include replication of the natural template M13 Gori by E . coli DNA polymerase III holoenzyme and the replication of a synthetic homopolymer by human immunodeficiency virus reverse transcriptase . A direct comparison of the fluorometric assay with the conventional isotopic assay shows that the fluorometric assay accurately reflects the extent of replication . By performing the assay reactions directly in 96-well plates and using a fluorescence plate reader to determine the extent of reaction, the time required to measure replication activities is significantly shortened.

Can J Microbiol, 1996 Oct, 42(10), 983 - 8
The amino acids of Escherichia coli enterotoxin B subunit involved in binding to Bio-Gel A-5m or to the glycoprotein from mouse intestinal epithelial cells; Kawase H et al.; We determined whether Arg13, Met31, and Ser95 of the heat-labile enterotoxin B subunit (LT-B) might be involved in Lt-B binding to oligosaccharides, which did not bind to the B subunit of the cholera toxin (CT-B) . Three LT-B mutants, R13H, M31L, and S95A were prepared by substituting three amino acid residues that differ in CT-B . These mutants formed a pentamer and exhibited the same binding ability to the GM1 ganglioside as native LT-B . Although these mutants did not bind to Bio-Gel A-5m, they did bind to the glycoprotein from mouse intestinal cells in the order R13H > M31L > S95A . These data suggest that Ser95, Met31, and Arg13 are important for LT-B binding to Bio-Gel A-5m, and that although Ser95 is also partially responsible for LT-B binding to the glycoprotein, Arg13 has no significant involvement in it.

Genetics, 1996 Oct, 144(2), 817 - 28
Sequence analysis of eukaryotic developmental proteins: ancient and novel domains; Mushegian AR et al.; Most of the genes involved in the development of multicellular eukaryotes encode large, multidomain proteins . To decipher the major trends in the evolution of these proteins and make functional predictions for uncharacterized domains, we applied a strategy of sequence database search that includes construction of specialized data sets and iterative subsequence masking . This computational approach allowed us to detect previously unnoticed but potentially important sequence similarities . Developmental gene products are enriched in predicted nonglobular regions as compared to unbiased sets of eukaryotic and bacterial proteins . Developmental genes that act intracellularly, primarily at the level of transcription regulation, typically code for proteins containing highly conserved DNA-binding domains, most of which appear to have evolved before the radiation of bacteria and eukaryotes . We identified bacterial homologues, namely a protein family that includes the Escherichia coli universal stress protein UspA, for the MADS-box transcription regulators previously described only in eukaryotes . We also show that the FUS6 family of eukaryotic proteins contains a putative DNA-binding domain related to bacterial helix-turn-helix transcription regulators . Developmental proteins that act extracellularly are less conserved and often do not have bacterial homologues . Nevertheless, several provocative similarities between different groups of such proteins were detected.

Biophys J, 1996 Oct, 71(4), 2213 - 21
Chaperonins GroEL and GroES: views from atomic force microscopy; Mou J et al.; The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM) . We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature . However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing . We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images . GroEL with GroES bound can be clearly distinguished from those without . The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL . Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers . These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM . They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.

Biophys J, 1996 Oct, 71(4), 2075 - 86
Interactions of Escherichia coli primary replicative helicase DnaB protein with nucleotide cofactors; Jezewska MJ et al.; Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides . The thermodynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of protein fluorescence and the degree of nucleotide binding . Fluorescence titrations using several MANT derivatives of nucleoside diphosphates (MANT-ADP, 3',2'-O-(N-methylantraniloyl)adenosine-5'-diphosphate; MANT-GDP, 3',2'-O(N-methylantraniloyl)guanosine-5'-diphosphate; MANT-CDP, 3',2'-O-(N-methylantraniloyl)cytidine-5'-diphosphate; MANT-UDP, 3',2'-O-(N-methylantraniloyl)uridine-5'-diphosphate) have shown that the DnaB helicase has a preference for purine nucleotides . Binding of all modified nucleotides is characterized by similar negative cooperativity, indicating that negative cooperative interactions are base-independent . Thermodynamic parameters for the interactions of the unmodified nucleotides (ADP, GDP, CDP, and UDP) and inorganic phosphate (P(i)) have been obtained by using the competition titration approach . To analyze multiple ligand binding to a finite circular lattice, for a general case in which each lattice binding site can exist in different multiple states, we developed a matrix method approach to derive analytical expressions for the partition function and the average degree of binding for such cases . Application of the theory to competition titrations has allowed us to extract the intrinsic binding constants and cooperativity parameters for all unmodified ligands . This is the first quantitative estimate of affinities and the mechanisms of binding of different unmodified nucleotides and inorganic phosphate for a hexameric helicase . The intrinsic affinities of all of the studied ATP analogs are lower than the intrinsic affinities of the corresponding ADP analogs . The implications of these results for the mechanism of helicase action are discussed.

J Gen Virol, 1996 Oct, 77 ( Pt 10), 2645 - 52
The nucleotide sequence and genomic organization of grapevine virus B; Saldarelli P et al.; Grapevine virus B (GVB) is a tentative member of the genus Trichovirus . The 5'-terminal region of the RNA genome of GVB comprises 5437 nucleotides and has been sequenced by the dideoxynucleotide chain termination method . Evidence was obtained that the RNA is capped . Two putative open reading frames (ORFs) were identified . ORF 1 coded for a 194.7 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses (i.e . methyltransferase, helicase and RNA-dependent RNA polymerase, in that order from the N to the C terminus) . ORF 2 encoded a 20 kDa polypeptide that did not show any significant sequence homology with protein sequences from the databases . The biological function of this polypeptide was not determined . Although the 20 kDa product was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and an antiserum produced, it could not be identified in GVB-infected plant tissue extracts . The GVB genome had the same size as that of apple chlorotic leaf spot virus (ACLSV), the type species of the genus Trichovirus, but differed substantially in the number (five compared to three), size and order of genes . Differences existed also in the extent of sequence homology between polymerases, which did not cluster together in tentative phylogenetic trees . The results of this study show that definitive and tentative trichovirus species differ molecularly to an extent that may warrant a taxonomic revision of the genus.

J Gen Virol, 1996 Oct, 77 ( Pt 10), 2441 - 5
Mother to fry, successful transfer of immunity against infectious haematopoietic necrosis virus infection in rainbow trout; Oshima S et al.; We have tested whether immunity can be transferred from a mother fish to its fry . Rainbow trout mother fish were inoculated against infectious haematopoietic necrosis virus (IHNV) by intraperitoneal injection of a fragment of the IHNV glycoprotein spanning amino acids 31 to 310 . This protein fragment was obtained by isolating the specific cDNA from Japanese IHNV strain HV7601 and expressing it in Escherichia coli . Fry from immunized and control fish were exposed to IHNV at various intervals after hatching, and their mortality monitored . Survival of the fry of immunized fish was significantly greater when exposure to virus occurred 7 days after hatching, and some immunity appeared to persist until at least 25 days after hatching.

Microbiology, 1996 Oct, 142 ( Pt 10), 2969 - 74
The thiJ locus and its relation to phosphorylation of hydroxymethylpyrimidine in Escherichia coli; Mizote T et al.; Extracts from Escherichia coli K-12 contained two distinct enzymes capable of catalysing the phosphorylation of hydroxymethylpyrimidine (HMP) to HMP monophosphate: pyridoxine kinase (EC 2.7.1.35) and an enzyme that has not previously been genetically analysed, HMP kinase (EC 2.7.1.49) . Two distinct genes, pdxL and thiJ, specify the activities of the former and latter enzymes, respectively . The inactivation of both genes by independent mutations in the same cell resulted in the complete loss of HMP kinase activity . Experiments with a series of strains that carry mutations in thiC, thiC pdxB, thiC pdxB pdxL and thiC pdxB pdxL thiJ revealed that the ability of the double mutant (pdxL thiJ) to utilize HMP in thiamin pyrophosphate biosynthesis was restored by introducing the wild-type allele corresponding to the thiJ mutation . The thiJ locus was mapped on the chromosome near the thiD and thiM loci, which govern the activities of phosphomethylpyrimidine kinase (EC 2.7.4.7) and hydroxyethylthiazole kinase (EC 2.7.1.50), respectively.

Microbiology, 1996 Oct, 142 ( Pt 10), 2759 - 66
P-fimbriae-producing septicaemic Escherichia coli from poultry possess fel-related gene clusters whereas pap-hybridizing P-fimbriae-negative strains have partial or divergent P fimbrial gene clusters; Dozois CM et al.; The organization of P fimbrial gene clusters of 13 papC-hybridizing Escherichia coli strains isolated from poultry with colisepticaemia, five P-fimbriae-expressing (P-positive) and eight P-fimbriae-non-expressing (P-negative), were examined by PCR and by Southern blot hybridization using primers or gene probes specific to the I, B, A, C or G genes . The absence of P fimbrial expression was associated with lack of PCR amplification of one or more of these genes, most commonly the I gene . Restriction endonuclease EcoRI, BamHI or PstI digests of genomic DNA from all strains hybridized with each of the gene probes and demonstrated polymorphisms between P-positive and P-negative strains . PstI digests of DNA from 12 of the 13 strains, when hybridized with the A gene probe, demonstrated a 0.1 kb fragment specific to the felA gene which encodes the major structural protein of F11 fimbriae . Hence, only the P-positive strains contained complete copies of fel-related gene clusters . In contrast, most of the pap-hybridizing P-negative strains contained partial or divergent P fimbrial gene clusters, which explains the lack of P fimbrial expression by these strains.

Plant Physiol, 1996 Oct, 112(2), 685 - 95
Both 5' and 3' sequences of maize adh1 mRNA are required for enhanced translation under low-oxygen conditions; Bailey-Serres J et al.; Alcohol dehydrogenase-1 (ADH1) synthesis in O2-deprived roots of maize (Zea mays L.) results from induced transcription and selective translation of ADH1 mRNA . The effect of ADH1 mRNA sequences on message stability and translation was studied in protoplasts of the maize cell line P3377.5' capped and 3' polyadenylated mRNA constructs containing the firefly gene (luc) for luciferase (LUC) or the Escherichia coli gene (uidA) for beta-glucuronidase (GUS) coding region were synthesized in vitro and electroporated into protoplasts that were cultured at 40 or 5% O2 . A LUC mRNA with a 17-nucleotide polylinker 5' untranslated region (UTR) was expressed 10-fold higher under aerobic conditions than under hypoxic conditions . Expression of five chimeric ADH1-GUS mRNAs was measured relative to this LUC mRNA . An mRNA containing the 5'-UTR and the first 18 codons of adh1 in a translational fusion with the GUS coding region and followed by the 3'-UTR of adh1 was expressed 57-fold higher at 5% O2 . Progressive deletion of adh1 5'-UTR and coding sequences reduced expression of the GUS-mRNA at 5% O2, but had little impact on expression of 40% O2 . Enhancement of expression in hypoxic protoplasts conferred by the adh1 5'-UTR and the first 26 codons decreased more than 3-fold when the adh1 3'-UTR was removed . In addition, the adh1 3'-UTR slightly inhibited expression in aerobic protoplasts . The physical half-lives of the GUS and LUC mRNAs were similar under both anaerobic and hypoxic conditions, indicating that expression levels were largely independent of mRNA stability . Thus, both adh1 5' and 3' mRNA sequences are required for enhanced translation in protoplasts under O2 deprivation.

Plant Physiol, 1996 Oct, 112(2), 669 - 75
Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii; Yildiz FH et al.; A Chlamydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli . E . coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity . The amino acid sequence of the C . reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus . ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions . Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.

Nucleic Acids Res, 1996 Oct 1, 24(19), 3763 - 70
Characterization of the DNA polymerase requirement of human base excision repair; Nealon K et al.; Base excision repair is one of the major mechanisms by which cells correct damaged DNA . We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template . In this report, we demonstrate the fractionation of a human cell extract into two required components . One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase beta (Polbeta) . Purified, recombinant Polbeta efficiently substituted for this fraction . Escherichia coli PolI, mammalian Poldelta and to a lesser extent Polalpha and epsilon also functioned in this assay . We provide evidence that multiple polymerases function in base excision repair in human cell extracts . A neutralizing antibody to Polbeta, which inhibited repair synthesis catalyzed by pure Polbeta by approximately 90%, only suppressed repair in crude extracts by a maximum of approximately 70% . An inhibitor of Polbeta, ddCTP, decreased base excision repair in crude extracts by approximately 50%, whereas the Polalpha/delta/epsilon inhibitor, aphidicolin, reduced the reaction by approximately 20% . A combination of these chemical inhibitors almost completely abolished repair synthesis . These data suggest that Polbeta is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.

Nucleic Acids Res, 1996 Oct 1, 24(19), 3748 - 55
A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries; Kayushin AL et al.; Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale . Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function . The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations . Purity of the target products has been confirmed by test reactions . The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach . A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.

Nucleic Acids Res, 1996 Oct 1, 24(19), 3714 - 21
Zn2+-sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation; Turner JS et al.; SmtB is a member of a family of repressors which dissociate from DNA in the presence of metals; Zn2+ being the most potent inducer of metallothionein gene (smtA) transcription in vivo . In Synechococcus PCC 7942 cells devoid of chromosomal smtB, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed lacZ expression driven by the smtA operator-promoter . Gel retardation assays with extracts from the complemented cells detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB . Elevated {Zn2+} alleviated repression in vivo by all of the mutants except H105R/H106R . These His residues (one or both) are therefore essential for Zn2+-sensing while, contrary to expectations, Cys residues are not . Hence different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the spectra of metals sensed . Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm that additional unknown factors form specific associations with the smt operator-promoter in elevated {Zn2+}.

Nucleic Acids Res, 1996 Oct 1, 24(19), 3670 - 6
The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation; Poot RA et al.; To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A18 mutation in this structure element . Previously, this mutation, which changes the central base pair of helix 2, C18--G917, to an A18xG917 mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested . Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits . Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control . However, the A18 mutation has a profound effect on the overall stability of the subunit . The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins . Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins . Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution . Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins . We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex . We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes.

Biochem J, 1996 Oct 1, 319 ( Pt 1), 33 - 8
Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells; Kuge O et al.; We have isolated a full-length cDNA clone of the Chinese hamster ovary (CHO) pssC gene, which encodes mitochondrial phosphatidylserine decarboxylase . The cDNA clone is capable of increasing phosphatidylserine decarboxylase activity to 11-fold in CHO-K1 cells . The pssC gene product predicted from the cDNA sequence is composed of 409 amino acid residues . In an in vitro translation system coupled with in vitro transcription, the cDNA clone directs the formation of a protein with an apparent molecular mass of 46 kDa . In CHO-K1 cells, the cDNA clone leads to the production of two major peptides with apparent molecular masses of 38 and 34 kDa, as determined by Western blotting with an antibody raised against a recombinant pssC protein . When CHO-K1 cells transfected with the cDNA clone are labelled with {35S}methionine for a short period, proteins immunoprecipitated with the antibody lack radioactive 38 and 34 kDa peptides, but contain two radioactive peptides with apparent molecular masses of 46 and 42 kDa instead . The pssC gene product predicted from the cDNA sequence has, near its C-terminus, a unique Leu-Gly-Ser-Thr sequence which is known as a processing site for Escherichia coli phosphatidylserine decarboxylase . A mutant pssC cDNA clone, in which Ser378 in the conserved sequence is replaced by Ala, leads to overproduction of 46, 42 and 38 kDa peptides, but not a 34 kDa peptide . This mutant clone is incapable of increasing phosphatidylserine decarboxylase activity, in contrast to the wild-type clone . These results indicate that the processing at the Leu-Gly-Ser-Thr sequence is essential for formation of the active enzyme . Thus, the pssC gene product is converted into mature phosphatidylserine decarboxylase through multiple steps of post-translational processing.

Biochem J, 1996 Oct 1, 319 ( Pt 1), 27 - 32
Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum; Witter K et al.; The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced . The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx . 26 kDa . Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium . PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum . The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively . Most of the sequence variation between species is located in the N-terminal part of the protein . The overall identity with the E . coli protein is markedly lower . The enzyme was expressed in E . coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E . coli . GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C . The Km value for GTP is 50 microM.

Biotechnol Appl Biochem, 1996 Oct, 24 ( Pt 2), 109 - 19
Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli; Ghag SK et al.; The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli . About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step . Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea . The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation . Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e . 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.

Mol Pharmacol, 1996 Oct, 50(4), 820 - 8
Vasopressin V2 receptor mutants that cause X-linked nephrogenic diabetes insipidus: analysis of expression, processing, and function; Oksche A et al.; We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus . COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific {3H}arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor . Membrane preparations from COS.M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type . By analyzing fusion proteins of the V2 receptor and Escherichia coli alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, W164S, S167L, and S167T lacked complex glycosylation and were expressed at low levels . The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded . In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant S167A were expressed in their mature form at wild-type levels; however, only the mutant S167A was functionally active . Site-directed mutagenesis of S167 revealed that elimination of the endogenous hydroxyl group (S167A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (S167T) and the replacement of the hydroxyl group by an isopropyl group (S167L) profoundly disturbed receptor processing . The data show that minute changes at codon 167 nearly abolish expression of a mature protein, thus defining structural requirements of this codon.

Virology, 1996 Oct 1, 224(1), 1 - 9
Temperature-sensitive mutations in the protein kinase-1 (pk-1) gene of the Autographa californica nuclear polyhedrosis virus that block very late gene expression; Fan X et al.; We have developed a genetic screen for temperature-sensitive mutations in the very late transcription apparatus of the Autographa californica nuclear polyhedrosis virus . This method starts with the BacPAKS virus, which has the Escherichia coli lacZ gene under the control of the very late polyhedrin promoter . The desired mutants are temperature-sensitive for beta-galactosidase production and can be complemented by wild-type virus, which lacks the lacZ gene . Two mutants created by nitrosoguanidine mutagenesis and identified by this screen, and one mutant identified by another screen, have been mapped by marker rescue to the viral protein kinase 1 gene (pk-1) . The protein kinase genes of these three mutant viruses have been sequenced, revealing the same point mutation in two of them and a different point mutation in the other . In each case, a single amino acid is changed: In two mutants, XF4 and XF5, Asp 92 is changed to Asn; in the other mutant, KT800, Thr 204 is changed to lle . Northern blotting of RNA made in cells infected by these three mutant viruses has shown that the accumulation of very late transcripts (lacZ and p10) is temperature-sensitive, but that accumulation of at least one late transcript (vp39) is not temperature-sensitive . Nuclear run-on transcription assays with two of the mutants indicate that very late transcription is somewhat temperature-sensitive, although this defect is not as pronounced as the temperature-sensitivity detected by Northern blotting . Transcription of at least one late gene (vp39) is not temperature-sensitive in cells infected by these two mutants . Thus, it appears that the viral protein kinase-1 is involved in very late gene expression . Some of this effect is at the transcription level, but some may also be exerted at the posttranscription level.

J Trauma, 1996 Oct, 41(4), 641 - 6
Cardiopulmonary effects of combined nitric oxide inhibition and inhaled nitric oxide in porcine endotoxic shock; Offner PJ et al.; BACKGROUND: Nitric oxide synthase (NOS) inhibition has been shown to potentiate lipopolysaccharide (LPS) associated pulmonary hypertension, which may worsen right ventricular (RV) dysfunction and decrease cardiac output during sepsis . This study evaluates whether inhaled nitric oxide can ameliorate the adverse cardiopulmonary effects of NOS inhibition during endotoxemia . METHODS: After an infusion of Escherichia coli LPS (200 micrograms/kg), animals were resuscitated with saline (1 mL/kg/min) and observed for 3 hours while mechanically ventilated (FIO2, 0.6; VT, 12 mL/kg; positive end-expiratory pressure, 5 cm H2O) . The LPS group (n = 6) received no additional treatment . The N-nitro-L-arginine methyl ester (NAME) group (n = 5) received L-NAME, a NOS inhibitor, 50 micrograms/kg/min for the last 2 hours . The NO+NAME group (n = 6) received inhaled NO (40 ppm) and L-NAME for the last 2 hours . The control group (n = 5) received only saline without LPS . Hemodynamic data and blood gases were collected hourly for 3 hours . RESULTS: L-NAME worsened LPS-associated pulmonary hypertension and RV dysfunction as reflected by decreased RV ejection fraction . Inhaled nitric oxide significantly decreased pulmonary hypertension and improved RV ejection fraction and stroke work index . There were no adverse systemic effects . CONCLUSIONS: Inhaled nitric oxide reverses pulmonary hypertension seen with L-NAME treatment during endotoxemia and may be a useful adjunct to NOS inhibition in the treatment of septic shock.

J Trauma, 1996 Oct, 41(4), 634 - 40
Role of platelet-activating factor antagonism in posthemorrhage septic shock in pigs; Abu-Zidan FM et al.; OBJECTIVES: To study the role of platelet-activating factor (PAF) antagonism in posthemorrhage septic shock in pigs . DESIGN: Experimental study . MATERIALS AND METHODS: Twelve anesthetized pigs were bled, kept with a mean arterial pressure of 30 mm Hg for 30 minutes, and then resuscitated with 50 mL/kg of isotonic saline . A continuous infusion of Escherichia coli endotoxin 36 micrograms/kg/hour was given intravenously for 3.5 hours starting 30 minutes after resuscitation . The animals were divided into two groups of six each . One group received I mg/kg of BB-882 (a potent specific PAF receptor antagonist) as a bolus during resuscitation, followed by a continuous infusion of BB-882 1 mg/kg/hour . The other group received vehicle alone . MEASUREMENTS AND MAIN RESULTS: The measured variables were blood temperature, heart rate, intravascular pressures, cardiac output, systemic and pulmonary vascular resistance, lung-thorax compliance, blood gases, hemoglobin oxygen saturation, packed cell volume and blood sugar and serum lactic acid concentrations . The group treated with BB-882 had significantly higher intracardiac pressures and cardiac output, and had less increase in systemic vascular resistance . The BB-882 group had significantly less lactic acidemia than the control group (p < 0.05, analysis of variance appropriate for repeated measurement design) . BB-882 had no effect on endotoxin-induced hypoxia or reduced lung-thorax compliance . CONCLUSIONS: PAF antagonism reduced the increase in systemic vascular resistance, improved cardiac output, and reduced lactic acidemia in posthemorrhage septic shock in pigs, but it did not improve hypoxia or reduced lung-thorax compliance.

J Clin Endocrinol Metab, 1996 Oct, 81(10), 3604 - 6
Hypercortisolemia increases plasma interleukin-10 concentrations during human endotoxemia--a clinical research center study; van der Poll T et al.; Hypercortisolemia directly before the administration of endotoxin (LPS) to normal humans completely prevents the release of the proinflammatory cytokine tumor necrosis factor, whereas hypercortisolemia 12 h to 7 days before the injection of LPS is associated with enhanced tumor necrosis factor release . To determine the effect of elevated cortisol levels on the secretion of the antiinflammatory cytokine interleukin-10 (IL-10), 23 healthy men were given iv LPS (lot EC-5; 2 ng/kg) alone or in combination with a continuous iv infusion of hydrocortisone (3 micrograms/kg.min) for 6 h immediately before or 6, 12, or 144 h before LPS injection . LPS induced a monophasic increase in plasma IL-10 concentrations that peaked after 2 h (162 +/- 27 pg/mL; P < 0.0001) . In subjects who were infused with hydrocortisone directly before LPS administration, IL-10 concentrations were much higher (1784 +/- 331 pg/mL; P < 0.0001 vs . LPS only), whereas hypercortisolemia 6, 12, or 144 h before LPS injection did not influence LPS-induced IL-10 levels . In human whole blood in vitro, hydrocortisone caused a dose-dependent reduction of LPS-induced IL-10 levels . Further, hydrocortisone reversed the increase in IL-10 concentrations by epinephrine in LPS-stimulated whole blood . Stimulation of IL-10 release may contribute to the antiinflammatory properties of glucocorticoids.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11268 - 73
Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress; Gidrol X et al.; Reactive oxygen species are common causes of cellular damages in all aerobic organisms . In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress . To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E . coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2 . Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family . The complemented mutant showed higher catalase activity . mRNA expression of the annexin gene in A . thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid . Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11149 - 54
Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain; Brown PK et al.; The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens . To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E . coli K-12 strain (chi)289 . The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E . coli chromosome . A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E . coli strains . To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E . coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence . The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens . Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens . One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype . The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map . Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11143 - 8
A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells; Su H et al.; Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections . The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported . The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells . The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C . The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor . Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding . Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP . These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells . Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae . Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11115 - 20
Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry; Blyn LB et al.; The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation . Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined . In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES . The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry . The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells . PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein . Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis . The closely related PCBP1 showed no stable interaction with the RNA . Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10673 - 8
The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange; Mazin AV et al.; The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule . Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences . The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA . Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site . We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition . The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange . We suggest that the secondary binding site has a dual role in DNA strand exchange . During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange . These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10632 - 7
Mutations in the C-terminal fragment of DnaK affecting peptide binding; Burkholder WF et al.; Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates . Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E . coli . This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK . Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening . The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate.

RNA, 1996 Oct, 2(10), 1011 - 21
In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function; Green R et al.; In vitro transcripts of Escherichia coli 23S rRNA are compromised severely (at least five orders of magnitude below natural 23S rRNA) in their ability to reconstitute into catalytically active, correctly assembled 50S subunits in a standard reconstitution procedure . Denaturation experiments suggest that this deficiency is the result of missing posttranscriptional modifications present in natural 23S rRNA . An in vitro complementation analysis was performed where partial natural 23S rRNA fragments prepared by RNase H digestion or hammerhead ribozyme cleavage were combined with the remaining RNA as a partial in vitro transcript in a standard reconstitution reaction and the peptidyl transferase activity was measured . This approach has identified a ca . 80-nt region in 23S rRNA (extending from position 2445 to 2523) containing the natural RNA element essential for E . coli 50S subunit assembly and has excluded the requirement for all but six of the known posttranscriptional modifications in 23S rRNA for 50S subunit assembly or peptidyl transferase activity . Importantly, this chimeric reconstitution approach provides a system for the analysis of pure mutant populations of 23S rRNA reconstituted into 50S subunits.

J Infect Dis, 1996 Oct, 174(4), 828 - 34
A protective domain of heat-shock protein 60 from Histoplasma capsulatum; Deepe GS Jr et al.; Vaccination with recombinant (r) hsp60 protects mice against Histoplasma capsulatum . To map the domain of rhsp60 that confers protection, four gene segments were cloned into pET19b and expressed in Escherichia coli . rhsp60 fragments were tested for their capacity to induce proliferation by sensitized splenocytes and to protect BALB/c and C57BL/6 mice . All polypeptides stimulated cells from BALB/c mice immunized with yeasts or with rhsp60 . Fragment 3 caused the most vigorous response by cells from mice immunized yeasts, and fragment 1 caused the most vigorous response by cells from mice immunized with rhsp60 . Splenocytes from yeast-immunized C57BL/6 mice did not recognize any polypeptide . In contrast, cells from C57BL/6 mice inoculated with rhsp60 responded to all fragments but most intensely to fragment 2 . In both strains, fragment 3 conferred protection against sublethal and lethal challenges . Thus, a common protective domain of hsp60 has been identified.

J Infect Dis, 1996 Oct, 174(4), 768 - 76
Colonization factors of enterotoxigenic Escherichia coli isolated from children in north India; Sommerfelt H et al.; Colonization factor antigens (CFAs) mediate attachment of enterotoxigenic Escherichia coli (ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhea . ETEC strains (n = 111) isolated from North Indian children from 1985 to 1989 were examined for CFAs and putative colonization factors (PCFs) . CFA/IV was the most common factor (26%), followed by coli surface antigen 17 (CS17) (19%), CFA/I (14%), PCFO166 (7%), and CFA/II (5%), while 24% of the isolates were negative for CFAs and PCFs . Among the strains producing heat-stable and heat-labile toxin (ST+LT+ strains), the STaI gene was strongly associated with the absence of known CFAs and PCFs, making the STaI+LT+ isolates an interesting target for the identification of previously undescribed factors . Repetitive sequence--based polymerase chain reaction revealed that the CS17+ strains, although clonally related, represented endemically circulating strains with a diversity greater than that of the CFA/I+ strains, which showed a substantial clonal clustering.

Biochemistry, 1996 Oct 1, 35(39), 12957 - 62
Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease; Wondrak EM et al.; The maturation of the human immunodeficiency virus type 1 protease (PR) from the Gag-Pol polyprotein is dependent on the intrinsic proteolytic activity of the dimeric Gag-Pol . Herein, we report the kinetics and conformational stabilities of two unique fusion proteins of the protease . In one, X28-PR, a random sequences of 28 amino acids (X28) was linked to the N terminus of the mature protease . In the second construct, X28-delta TF*PR*delta Pol, X28 is fused to the protease which is flanked at both its termini by short sequences (delta) which correspond to the native sequences of the Gag-Pol precursor . Autoprocessing of the latter protein was prevented by inserting an Ala at the native protease cleavage sites . The measured kinetic parameters and the pH-rate profile of both enzymes are nearly identical to those of the mature protease . However, these fusion proteins are more sensitive to acid and urea denaturation than the mature protease . The decrease in the conformational stability of X28-PR and X28-delta TF*PR*delta Pol is reflected by increases in their apparent dissociation constants (Kd) from < 5 nM to approximately 180 and 25 nM, respectively . These results suggest that subunit interactions and hence the dimer stability of the protease domain in the Gag-Pol polyprotein differ from those of the mature protease . The high Kd of X28-PR further suggests that addition of non-native sequences to the N terminus of the protease destablizes the dimer.

Biochemistry, 1996 Oct 1, 35(39), 12926 - 32
Base analog and neighboring base effects on substrate specificity of recombinant human G:T mismatch-specific thymine DNA-glycosylase; Sibghat-Ullah et al.; We studied the substrate specificity of the human G:T mismatch-specific thymine glycosylase that initiates the repair of G:T and G:U base mismatches to G:C base pairs . Such mismatches arise when 5-methylcytosine or cytosine deaminate spontaneously (and hydrolytically) in DNA . Substrates were 45-bp DNA heteroduplexes that bore single G:T, m6G:T, 2,6-diaminopurine:T, 2-amino-6-(methylamino)-purine:T, 2-aminopurine:T, and G:m4T mispairs . The bases 5' to the poorly matched G were altered in selected G:T substrates to yield mispairs in four different contexts, ApG, CpG, GpG, and TpG . The recombinant thymine glycosylase was incubated with the 45-bp DNA substrates, each labeled at the 5'-terminus of the strand containing the mismatched T . The DNAs were then treated with 0.1 N NaOH to catalyze phosphodiester bond breakage at the newly-generated AP sites, and the products were analyzed on DNA sequencing gels . As indicated by the amounts of the 20-nt incision product, the removal of the thymine base by the enzyme increased linearly between 0 and 40 min at which time the generation of product from all substrates ceased, probably because of enzyme inactivation . The rate of incision was greatest (0.7 fmol/min) with DNA containing the G:T mispair followed by the DNA containing the m6G:T mispair (0.38 fmol/min) and the DNA with the 2-amino-6-(methylamino)purine:T mispair (0.15 fmol/ min); the extent of reaction was 90%, 40%, and 20% respectively . By contrast to previous findings with cell-free extracts, DNA substrates containing 2,6-diaminopurine:T, 2-aminopurine:T, and G:m4T mispairs were not incised (< 2%) . The amount of incision of the 45-bp DNA substrates containing G:T mispairs in the CpG context was 3-12-fold greater than in the TpG, GpG, and ApG contexts.

Biochemistry, 1996 Oct 1, 35(39), 12919 - 25
Kinetic mechanism of the 3'-->5' proofreading exonuclease of DNA polymerase III . Analysis by steady state and pre-steady state methods; Miller H et al.; DNA polymerase III holoenzyme is the major replicative enzyme in Escherichia coli . An important component of the high-fidelity DNA synthesis that is characteristic of DNA polymerase III holoenzyme is the 3'-->5' proofreading exonuclease activity resident in the epsilon subunit . Steady state and pre-steady state conditions have been used to determine equilibrium and Michaelis constants for substrate binding and the rate constant for cleavage by purified epsilon subunit . The steady state kinetic constants are K(m) = 16 +/- 6 microM and kcat = 210 +/- 23 s-1 for degradation of single-stranded DNA by epsilon . These steady state values are in agreement with the rate constants determined for excision of the 3' nucleotide of a dT10 oligomer under pre-steady state conditions . Using a simple two-step model, E + Dn reversible E.Dn-->E + Dn-1, we find K = 12 microM and kf = 280 s-1 for the dT10 substrate . In these experiments, epsilon subunit acts in a distributive manner and product release is not the rate-limiting step . Activity of the epsilon subunit on paired DNA oligonucleotides with zero to three mismatches at the 3' terminus indicates that an additional step is required in the mechanism . In the scheme Dn reversible Dn* + E reversible E.Dn*-->E + Dn-1, the 3' terminus undergoes a conformational change or "melts" before the DNA is a substrate for epsilon subunit . With this additional step, the values for binding of activated substrate and cleavage are the same as those for single-stranded DNA . The kinetics for exonucleolytic degradation of single-stranded, paired, and mispaired oligonucleotides support the model that the rate-limiting step in exonucleolytic proofreading of DNA by epsilon subunit is the DNA-melting step.

Biochemistry, 1996 Oct 1, 35(39), 12915 - 8
Site-directed spin labeling demonstrates that transmembrane domain XII in the lactose permease of Escherichia coli is an alpha-helix; Voss J et al.; Functional lactose permease mutants containing single-Cys residues at positions 387-402 {He, M . M., Sun, J., & Kaback, H . R . (1996) Biochemistry 35, 12909-12914} and a biotin acceptor domain in the middle cytoplasmic loop were solubilized in n-dodecyl-beta-D-maltopyranoside and purified by avidin affinity chromatography . Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy . Analysis of the electron paramagnetic resonance spectral line shapes and the influence of O2 on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to O2, respectively . The data show a periodic dependence of both mobility and accessibility on sequence position consistent with an alpha-helical structure . These results provide direct support for the contention that transmembrane domain XII is in an alpha-helical conformation and on the periphery of the 12-helix bundle that comprises the lactose permease molecule.

Biochemistry, 1996 Oct 1, 35(39), 12909 - 14
Cysteine-scanning mutagenesis of transmembrane domain XII and the flanking periplasmic loop in the lactose permease of EScherichia coli; He MM et al.; Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys . Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377-->Cys and Leu385-->Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400-->Cys exhibits < 20% of C-less activity . Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377-->Cys and Leu385-->Cys which are expressed about 40% as well as C-less and mutant Leu400-->Cys which is hardly detectable . When transferred to the wild-type background, however, mutant Leu400-->Cys is expressed normally and exhibits highly significant transport activity . Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent . Mutants Val367-->Cys, Gly370-->Cys, and Tyr373-->Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated . The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373 . In the following paper {Voss, J., He, M . M., Hubbell, W . L., & Kaback, H . R . (1996) Biochemistry 35, 12915-12918}, site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an alpha-helical conformation.

Biochemistry, 1996 Oct 1, 35(39), 12901 - 8
Relation between the oligomerization state and the transport and phosphorylation function of the Escherichia coli mannitol transport protein: interaction between mannitol-specific enzyme II monomers studied by complementation of inactive site-directed mutants; Boer H et al.; Previous experiments with the mannitol-specific enzyme II of Escherichia coli, EIImtl, have demonstrated that (1) the enzyme is a dimer, (2) the dimer is necessary for maximum activity, and (3) phosphoryl groups could be transferred between EIImtl subunits {van Weeghel et al . (1991) Biochemistry 30, 1768-1773; Weng et al . (1992) J . Biol . Chem . 267, 19529-19535; Weng & Jacobson (1993) Biochemistry 32, 11211-11216; Stolz et al . (1993) J . Biol . Chem . 268, 27094-27099} . The experiments in this article address the mechanistic role of the dimer . They indicate that the A, B, and C domains of EIImtl preferentially interact within the same subunit . Site-directed mutants in each of the three domains of EIImtl were used to study phosphoryl group transfer by the EIImtl dimer in vitro and mannitol transport in vivo . The C domain mutant, EIImtl-G196D, which was unable to bind mannitol, and the separated C domain, IICmtl, which was unable to phosphorylate mannitol, formed a heterodimer which was capable of mannitol phosphorylation in vitro and mannitol transport in vivo . The rates of phosphorylation were approximately 10-fold lower in heterodimers containing two inactive subunits relative to the rates in heterodimers containing one inactive and one wild type subunit; phosphoryl group transfer through one subunit is kinetically preferred to intersubunit transfer . Heterodimers formed in vivo between one wild type EIImtl subunit and the CB domain double mutant, EIImtl-G196D/C384S, transported mannitol as rapidly as wild type EIImtl alone; the presence of the inactive double mutant subunit did not reduce the transport rate . Thus, only one active A, B, and C domain in the dimer is sufficient for transport and phosphorylation activity, and if all three domains are situated on the same subunit, maximum rates are achieved.

Biochemistry, 1996 Oct 1, 35(39), 12788 - 95
Rescue of the horseradish peroxidase His-170-->Ala mutant activity by imidazole: importance of proximal ligand tethering; Newmyer SL et al.; The proximal iron ligand in horseradish peroxidase (HRP) is His-170 . The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized . At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm . Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron . Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM . Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group . Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im . The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS . The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im . The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im . The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1 . The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation . However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant . Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine . In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.

Biochemistry, 1996 Oct 1, 35(39), 12742 - 61
Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity; Pelletier H et al.; Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain . In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP {Pelletier, H., Sawaya, M . R., Kumar, A., Wilson, S . H., & Kraut, J . (1994) Science 264, 1891-1903} . The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations . The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies . Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain . DNA binding by both HhH motifs is facilitated by a metal ion . In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins . The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes . These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities . A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.

Biochemistry, 1996 Oct 1, 35(39), 12677 - 85
Cysteine scanning mutagenesis at 40 of 76 positions in villin headpiece maps the F-actin binding site and structural features of the domain; Doering DS et al.; Villin headpiece, the 76 amino acid, C-terminal domain of villin, is one of the two F-actin binding sites in villin necessary for F-actin bundling activity . Expression and study of recombinant headpiece revealed the domain to be remarkably thermostable (Tm = 74 degrees C) for a non-disulfide-bonded domain . Forty independent point mutations to cysteine of headpiece have been purified and tested for their actin binding activity, cysteine reactivity, and thermal stability . These assays identify two segments of headpiece, near amino acids 38 and 70 of headpiece, in which mutations to cysteine significantly disrupt cosedimentation of headpiece with F-actin . Assay of the thermal stability of these mutants and assay of the reactivity of the introduced cysteine show that these amino acids are mutations at the protein surface that do not perturb the overall structure of the domain . The actin binding mutants are replacements to cysteine of Lys38, Glu39, Lys65, Lys70, Lys71, Leu75, and Phe76 of headpiece . We propose that these discontinuous segments of charged amino acids define the F-actin binding contacts of the headpiece domain . The assay of mutants for effects on the thermal stability of helical structure as well as the assay of reactivity of the introduced sulfhydryl group identify candidate positions that are involved in the stabilizing core and internal structure of the domain . The cysteine scanning mutagenesis also identifies an amino-terminal subdomain (Val1-Leu35) and a predominantly helical carboxy-terminal subdomain (Pro36-Phe76).

Biochemistry, 1996 Oct 1, 35(39), 12659 - 70
Solution conformation of the N-(deoxyguanosin-8-yl)-1-aminopyrene ({AP}dG) adduct opposite dC in a DNA duplex; Mao B et al.; Combined NMR-molecular mechanics computational studies were undertaken on the C8-deoxyguanosine adduct formed by the carcinogen 1-nitropyrene embedded in the d(C5-{AP}G6-C7).d(G16-C17-G18) sequence context in a 11-mer duplex, with dC opposite the modified deoxyguanosine . The exchangeable and nonexchangeable protons of the aminopyrene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution . There was a general broadening of several proton resonances for the three nucleotide d(G16-C17-G18) segment positioned opposite the {AP}dG6 lesion site resulting in weaker NOEs involving these protons in the adduct duplex . The solution conformation of the {AP}dG.dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space . The aminopyrene ring of {AP}dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs . The modified deoxyguanosine ring of {AP}dG6 is displaced into the major groove and stacks with the major groove edge of dC5 in the adduct duplex . Both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue are consistent with a syn glycosidic torsion angle for the {AP}dG6 residue . The dC17 base on the partner strand is displaced from the center of the helix toward the major groove as a consequence of the aminopyrene ring intercalation into the helix . This base-displaced intercalative structure of the {AP}dG.dC 11-mer duplex exhibits several unusually shifted proton resonances which can be accounted for by the ring current contributions of the deoxyguanosinyl and pyrenyl rings of the {AP}dG6 adduct . In summary, intercalation of the aminopyrene moiety is accompanied by displacement of both {AP}dG6 and the partner dC17 into the major groove in the {AP}dG.dC 11-mer duplex.

Arch Biochem Biophys, 1996 Oct 1, 334(1), 158 - 62
Failure of tumor necrosis factor and interleukin-1 to elicit superoxide production in the mitochondrial matrices of mammalian cells; Gardner PR et al.; Subversion of mitochondrial electron transport to the production of O2.- has been proposed as a mechanism of tumor necrosis factor (TNF)-mediated cell killing and to a lesser extent interleukin-1 (IL-1) and lipopolysaccharide (LPS) cytotoxicity . We utilized the O2.- -sensitive aconitases to measure changes in steady-state 02.- levels in the mitochondrial matrix and cytoplasm of cultured mammalian cells in response to these inflammatory mediators . TNF alpha did not measurably affect aconitase activity, and thus mitochondrial 02.- production, in either cultured human A549 cells or murine L929 cells while TNF alpha clearly caused cytotoxicity as revealed by impaired mitochondrial respiration . IL-1 alpha and Escherichia coli LPS also failed to affect the aconitase activity in A549 cells . Neither the O2.- scavenger Mn(III) TMPyP nor the H2O2 scavenger catalase protected L929 cells against the cytotoxicity of TNF alpha . In conclusion, TNF, IL-1, and LPS do not appear to exert cytotoxicity, or MnSOD gene induction effects, by eliciting mitochondrial O2.- production.

Arch Biochem Biophys, 1996 Oct 1, 334(1), 83 - 8
Effect of activation of protein phosphatase 1 on sulfhydryl reactivity; Chu Y et al.; Myofibril protein phosphatase 1 (PP1) from bovine heart, identified as PP1alpha, was purified in a latent form which was dependent on Co2+ or Mn2+ for activity (Y . Chu, S . E . Wilson, and K . K . Schlender (1994) Biochim . Biophys . Acta 1208, 45-54) . This was also true for recombinant PP1 alpha expressed in Escherichia coli (Z . Zhang, G . Bai, S . Deans-Zirattu, M . F . Browner, and E . Y . C . Lee (1992) J . Biol . Chem . 267, 1484-1490) . Here we report on the change in the sulfhydryl reactivity during the cation activation process . The activation of myofibrillar PP1 by Co2+ was prevented by 10 mM dithiothreitol (DTT) and incubation of the Co2+-activated enzyme with 50 mM DTT reversed the activation . Activation of recombinant PP1alpha was associated with 57Co2+ incorporation into PP1 . DTT reversal of Co2+-activated PP1 was accompanied by release of Co2+ from the enzyme . The latent PP1 modified with 2-nitro-5-thiocyanobenzoic acid (NTCB) or N-ethylmaleimide (NEM) did not bind Co2+ and could not be activated by Co2+ . Conversely, the Co2+-activated PP1 was resistant to inactivation with NTCB and less sensitive to NEM . Similarly, PP1 pretreated with NTCB was not activated by Mn2+ and the Mn2+-activated enzyme was also resistant to NTCB inhibition . The number of sulfhydryls of nondenatured PP1, reactive with 5, 5'-dithiobis{2-nitrobenzoic acid} (DTNB), was reduced from approximately 8 to 2-3 mol/mol when the enzyme was activated with Co2+ or Mn2+ . After denaturation with guanidine-HCl, the number of reactive sulfhydryls of nonactivated PP1 and Co2+-activated PP1 was approximately 10 mol/mol enzyme . These results suggest that when PP1 is activated by Co2+ or Mn2+, the enzyme undergoes a conformational change resulting in some of the cysteine sulfhydryls no longer being accessible to chemical modification.

Arch Biochem Biophys, 1996 Oct 1, 334(1), 37 - 42
The H385N mutant of 5-enolpyruvylshikimate-3-phosphate synthase: kinetics, fluorescence, and nuclear magnetic resonance studies; Shuttleworth WA et al.; The site-directed mutagenesis of histidine-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported . The H385N mutant is compared with wild type by a number of methods . H385N was found to retain 6% activity . Kinetic parameters, including Km values for the natural substrates and Ki and Kd values for the inhibitor glyphosate, were found to be similar to wild type . Unlike wild-type enzyme, H385N EPSP synthase does not show accumulation of enzyme-bound product (EPSP) in the 13C NMR spectrum under equilibrium conditions . These results suggest that this H385N mutant is less catalytically competent than the previously studied H385Q mutant and that the N(epsilon)H of histidine may be involved in hydrogen bonding to another residue involved in complexing the substrates/ products.

Arch Biochem Biophys, 1996 Oct 1, 334(1), 27 - 36
Molecular characterization and resistance to hydrogen peroxide of two fructose-1,6-bisphosphatases from Synechococcus PCC 7942; Tamoi M et al.; In Synechococcus PCC 7942 cells, two catalytic fructose-1,6-bisphosphatase isoenzymes, designated F-I and F-II, have been resolved by chromatography on a HiLoad 26/10 Q Sepharose column at 0.24 and 0.34 M of NaCl, respectively; the former represented the major part of the total extractable enzyme activity . F-I has been purified to electrophoretic homogeneity from the cells . F-I and F-II had respective molecular masses of 160 and 150 kDa and each enzyme was composed of four identical subunits . F-I hydrolyzed both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, whereas F-II hydrolyzed only fructose 1,6-bisphosphate . The apparent Km values of F-I and F-II for fructose 1,6-bisphosphate were 52 +/- 4.5 and 25 +/- 1.5 microM, respectively . F-I was inhibited by AMP with a Ki value of 0.26 mM, but F-II was not affected by AMP . The F-I failed to cross-react by Western blotting with the antibody raised against F-II; similarly, the F-II did not react with the F-I antibody . The genes encoding F-I and F-II were cloned from the chromosomal DNA of Synechococcus PCC 7942 . A 1068-bp open reading frame, encoding F-I of 356 amino acid residues (approx molecular mass of 38.3 kDa) was observed . The nucleotide sequence of the F-II gene showed an open reading frame of 1017 bp that encodes a protein of 339 amino acid residues (approx molecular mass of 37.2 kDa) . The recombinant enzymes expressed in Escherichia coli as well as the native enzymes of F-I and F-II from Synechococcus PCC 7942 cells were resistant to 1 mM hydrogen peroxide unlike the light-activated higher plant chloroplast enzymes.

J Am Vet Med Assoc, 1996 Oct 1, 209(7), 1291 - 3
Effect of vaccination with an Escherichia coli bacterin-toxoid on milk production in dairy cattle; Musser JM et al.; OBJECTIVE: To determine whether vaccination of lactating cattle with an Escherichia coli J5 bacterin-toxoid would produce a significant short-term change in milk production . DESIGN: Randomized, controlled clinical trial . ANIMALS: 84 healthy, lactating cows (42 Holsteins and 42 Jerseys) . PROCEDURE: Control and vaccinated cows were paired on the basis of breed, days in milk, daily milk production 1 week prior to vaccination, and parity . One cow in each pair was inoculated IM with a commercially available bacterin-toxoid according to label directions; the other cow was given saline solution . Cows were milked twice daily for 5 days before and 5 days after inoculation . Milk production was compared by ANCOVA . RESULTS: Vaccinated cows produced significantly less milk than did control cows at the second and third milkings after inoculation . At these milkings, milk production in vaccinated cows was approximately 7% less than that of controls . CLINICAL IMPLICATIONS: Vaccination of lactating cattle with an E coli J5 bacterin-toxoid may cause a significant short-term decrease in milk production.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 253 - 8
Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil; Nishimura LS et al.; The electrophoretic profiles of outer membrane proteins and lipopolysaccharide of sixty-five enterotoxigenic Escherichia coli of different serotypes and virulence-associated factors, toxin and colonization factors were determined . A close relationship between serotype and outer membrane protein and lipopolysaccharide patterns could be observed . No correlation could be found between the electrophoretic profiles and the expression of virulence-associated factors . The observed homogeneity of outer membrane protein and lipopolysaccharide profiles suggested the presence of only a few clones in the samples studied, and supported the use of outer membrane protein and lipopolysaccharide analysis as a useful epidemiological tool in the characterization of enterotoxigenic Escherichia coli isolates.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 247 - 52
Characterization and sequence of the Escherichia coli panBCD gene cluster; Merkel WK et al.; A 4589 bp DNA segment containing the Escherichia coli panBCD gene cluster was sequenced, and found to contain 6 complete open reading frames . panB, panC, and panD were identified by subcloning and insertional mutagenesis . The orientation of panD was also confirmed by orientation-specific expression of asparate-1-decarboxylase . panB and panC lie adjacent to one another, but are separated from panD by orf3, which is oriented in the opposite direction . Interruptions in the remaining open reading frames did not affect growth on glucose-minimal medium . No significant similarity to sequences in databases was found for orf1 and orf2 . Orf3 contained extensive similarity to reading frames defined by E . coli yjiP, yjiQ, yhgA, and yafD . The function of these amino acid sequences is as yet undefined.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 217 - 21
Isolation and characterization of hemolytic genes from Actinobacillus actinomycetemcomitans; Kato T et al.; Periodontopathic Actinobacillus actinomyceremcomitans produces hemolysin and other leukotoxins . In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A . actinomycetemcomitans ATCC 43718 on blood agar plates . DNA hybridization analysis indicates that there were two distinct hemolytic genes present . Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit . Rabbit antiserum to A . actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 211 - 6
Carboxyl terminal region of the MukB protein in Escherichia coli is essential for DNA binding activity; Saleh AZ et al.; The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity . We have isolated a mutation, mukB1013, causing a substitution of valine at position 1379 to leucine . This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected . A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 175 - 83
Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum; Garcia B et al.; A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-alpha-glucan hydrolase, EC 3.2.1.11) was isolated and characterized . cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization . Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs . The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system . The expression of a 2 kb cDNA fragment in E . coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody . The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide . There was 29% sequence identity between the P . minioluteum dextranase and the dextranase from Arthrobacter sp . CB-8.

Biometals, 1996 Oct, 9(4), 327 - 35
DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases; Dowjat WK et al.; Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress . However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell . To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied . High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms . Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme . The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD . Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2 . Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide . It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA . Both types of SOD were shown to effectively cleave RNA . These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.

Nat Struct Biol, 1996 Oct, 3(10), 856 - 62
Novel active site in Escherichia coli fructose 1,6-bisphosphate aldolase; Blom NS et al.; The molecular architecture of the Class II E . coli fructose 1,6-bisphosphate aldolase dimer was determined to 1.6 A resolution . The subunit fold corresponds to a singly wound alpha/beta-barrel with an active site located on the beta-barrel carboxyl side of each subunit . In each subunit there are two mutually exclusive zinc metal ion binding sites, 3.2 A apart; the exclusivity is mediated by a conformational transition involving side-chain rotations by chelating histidine residues . A binding site for K+ and NH4+ activators was found near the beta-barrel centre . Although Class I and Class II aldolases catalyse identical reactions, their active sites do not share common amino acid residues, are structurally dissimilar, and from sequence comparisons appear to be evolutionary distinct.

J Clin Invest, 1996 Oct 1, 98(7), 1550 - 9
Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: role on diaphragmatic contractile dysfunction; Boczkowski J et al.; Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines . The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction . Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation . 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers . Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles . Diaphragmatic force was significantly decreased 12 and 24 h after LPS . This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine . We conclude that iNOS was induced in the diaphragm after E . Coli LPS inoculation in rats, being involved in the decreased muscular force.

J Bacteriol, 1996 Oct, 178(20), 6082 - 6
Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system; Hoischen C et al.; Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding . The results substantiate the conclusion that regulation of the lactose permease in E . coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.

J Bacteriol, 1996 Oct, 178(20), 6006 - 12
Fis binding in the dnaA operon promoter region; Froelich JM et al.; The region between the rpmH and dnaA genes contains five promoters that divergently express the ribosomal protein L34 and the proteins of the dnaA operon, including DnaA, the beta clamp of DNA polymerase III holoenzyme, and RecF . The DNA-binding protein Fis was shown by the band shift assay to bind near the rpmHp2 and dnaAp2 promoters and by DNase I footprinting to bind to a single site in the dnaAp2 promoter overlapping the -35 and spacer sequences . There were no observable differences in Fis affinity or the angle of bending induced by Fis between methylated and unmethylated DNA fragments containing the Fis binding site in the dnaAp2 promoter . Fis directly or indirectly represses the expression of DnaA protein and the beta clamp of DNA polymerase III . A fis null mutant containing a dnaA-lacZ in-frame fusion had twofold greater beta-galactosidase activity than a fis wild-type strain, and induced expression of Fis eliminated the increase in activity of the fusion protein . A two- to threefold increase in the levels of DnaA and beta clamp proteins was found in a fis null mutant by immunoblot gel analysis.

J Bacteriol, 1996 Oct, 178(20), 5989 - 94
Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation; Kaminski PA et al.; Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations . To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed . As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles . In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates . In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity . Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation . In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities . In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels . Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth . Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

J Bacteriol, 1996 Oct, 178(20), 5971 - 6
"Division potential" in Escherichia coli; Donachie WD et al.; The phenotype of a minC mutant has been reexamined and found to correspond closely to the quantitative predictions of Teather et al . (R . M . Teather, J . F . Collins, and W . D . Donachie, J . Bacteriol . 118:407-413, 1974) . We confirm that the number of septa formed per generation per cell length is fixed and independent of the number of available division sites and that "division potential" is directly proportional to cell length . In the minC mutant, septa form with equal probabilities at cell poles, cell centers, and cell quarters . In addition, we show that the time to next division is inversely related to cell length while division is asynchronous in long cells, suggesting that a single cell can form only one septum at a time.

J Bacteriol, 1996 Oct, 178(20), 5954 - 9
Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants; Francetic O et al.; Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway . An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export . The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence . Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined . Most of the mutations caused severe defects in RBP export . In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB . Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed . Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export . However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB . These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.

J Bacteriol, 1996 Oct, 178(20), 5925 - 9
The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation; Kolmar H et al.; DegP and DegQ are homologous endoproteases found in the periplasmic compartment of Escherichia coli . The studies presented here suggest that DegP and DegQ have very similar substrate specificities and cleave substrates which are transiently or globally denatured . Model substrates were cleaved at discrete Val/Xaa or Ile/Xaa sites, suggesting that aliphatic, beta-branched residues, which are typically buried in the hydrophobic core of most proteins, are important determinants of cleavage specificity . Indeed, the peptide bonds cleaved in the model substrates are generally inaccessible in the native three-dimensional structures . In addition, a chimeric fusion protein, which is a DegP substrate in vivo, is degraded in vitro only after reduction of its intramolecular disulfide bonds . Taken together, these findings suggest that DegP and DegQ may degrade transiently denatured proteins, unfolded proteins which accumulate in the periplasm following heat shock or other stress conditions, and/or newly secreted proteins prior to folding and disulfide bond formation . Cross-linking studies indicate that both DegP and DegQ form dodecamers in solution and thus are similar to many other intracellular proteases which form large oligomeric complexes.

Endocrinology, 1996 Oct, 137(10), 4308 - 15
Recombinant human growth hormone (GH)-binding protein enhances the growth-promoting activity of human GH in the rat; Clark RG et al.; To address the role of the GH-binding protein (GHBP) in GH physiology, two forms of recombinant human GHBP (rhGHBP) were given alone or in combination with rhGH to hypophysectomized rats or GH-deficient dwarf rats . Hypophysectomized rats were given daily sc injections of excipient, hGH, rhGHBP, or rhGH plus rhGHBP (produced in Escherichia coli) for 7 days . Injections of rhGH induced dose-related body weight gain and bone growth that were increased by the coadministration of rhGHBP with rhGH; rhGHBP alone had no effect . Serum insulin-like growth factor increased 24 h later when rhGH was given together with rhGHBP (P < 0.01), but not when rhGH was given alone . E . coli-derived rhGHBP also enhanced the bioactivity of coadministered rhGH in the GH-deficient dwarf rat . In contrast, the glycosylated rhGHBP, made in human A293 cells, inhibited the growth-promoting activity of coadministered rhGH . The opposite effects of these two forms of rhGHBP could be explained by clearance studies that showed radiolabeled rhGH bound to A293 cell-derived rhGHBP to be cleared more rapidly from the blood than free rhGH . Natural rabbit GHBP and E . coli-derived rhGHBP both prolonged the presence of rhGH in blood . It is proposed that by slowing the clearance of GH, GHBP increased the bioactivity of GH . In summary, codelivery of rhGHBP and rhGH caused a dose-dependent enhancement of the activity of rhGH in two rat models of GH deficiency . This suggests that endogenous circulating GHBP may increase the activity of blood-borne GH in a similar manner.

J Bacteriol, 1996 Oct, 178(19), 5813 - 6
Conjugative transposon Tn916: evidence for excision with formation of 5'-protruding termini; Manganelli R et al.; Conjugative transposons are genetic elements able to promote their own intracellular transposition and intercellular conjugal transfer . They move by an excision-integration system related to that of lambdoid phages, in which the first step is the excision of the transposon from the donor replicon to form a covalently closed circular intermediate which contains a heteroduplex joint . In this work, sequencing both strands of the circular intermediate heteroduplex joint, it was found that, as during lambda phage excision, Tn916 excises from the host DNA by 5'-protruding staggered endonucleolytic cleavages.

J Bacteriol, 1996 Oct, 178(19), 5803 - 5
Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli; Fralick JA; A study examining the influence of TolC on AcrA, AcrR, and MarR1 mutants indicates that functional TolC is required for the operation of the AcrAB efflux system and for the expression of the Mar phenotype . That the effect of TolC on the AcrAB pump is not regulatory in nature is shown by studies measuring the influence of a tolC::Tn10 insertion mutation on the expression of an acrA::lacZ reporter fusion . These results are compatible with the hypothesis that TolC is a component of the AcrAB efflux complex.

J Bacteriol, 1996 Oct, 178(19), 5797 - 802
Molecular, genetic, and biochemical characterization of the serC gene of Methanosarcina barkeri Fusaro; Metcalf WW et al.; The Methanosarcina barkeri serC gene, encoding phosphoserine aminotransferase, was cloned by complementation of an Escherichia coli serC mutant, and its nucleotide sequence was determined . The M . barkeri SerC protein shares significant homology with other known SerC proteins . E . coli serC hosts carrying the cloned gene express phosphoserine aminotransferase activity, verifying the function of this gene.

J Bacteriol, 1996 Oct, 178(19), 5790 - 2
The nucleoid protein H-NS facilitates chromosome DNA replication in Escherichia coli dnaA mutants; Katayama T et al.; Growth inhibition of the dnaA(Cs) mutant, which overinitiates chromosome replication, was shown to be dependent upon the nucleoid protein H-NS . {3H}thymine incorporation experiments indicated that the absence of H-NS inhibited overreplication by the dnaA(Cs) mutant . In addition, the temperature-sensitive phenotype of a dnaA46 mutant was enhanced by disruption of H-NS . These observations suggest that H-NS directly or indirectly facilitates the initiation of chromosome replication.

J Bacteriol, 1996 Oct, 178(19), 5787 - 9
Effect of traY amber mutations on F-plasmid traY promoter activity in vivo; Silverman PM et al.; We have examined the effect of the F plasmid TraY protein on tra gene expression in vivo . Expression was assayed as alkaline phosphatase activity in cells containing a traY phi(traA'-'phoA)hyb operon under traY promoter control . Amber mutations in traY significantly reduced alkaline phosphatase activity . Since nonsense polarity effects were minimal, if they occurred at all, these data provide the first direct evidence that TraY regulates tra gene expression.

J Bacteriol, 1996 Oct, 178(19), 5781 - 6
Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme; Sakurada K et al.; We have cloned a gene (aphA) encoding acetylpolyamine amidohydrolase from Mycoplana ramosa ATCC 49678, (previously named Mycoplana bullata) . A genomic library of M . ramosa was screened with an oligonucleotide probe designed from a N-terminal amino acid sequence of the enzyme purified from M . ramosa . Nucleotide sequence analysis revealed an open reading frame of 1,023 bp which encodes a polypeptide with a molecular mass of 36,337 Da . This is the first report of the structure of acetylpolyamine amidohydrolase . The aphA gene was subcloned under the control of the trc promoter and was expressed in Escherichia coli MM294 . The recombinant enzyme was purified, and the enzymatic properties were characterized . Substrate specificities, Km values, and Vmax values were identical to those of the native enzyme purified from M . ramosa . In the analysis of the metal-substituted enzymes, we found that the acid limb of pH rate profiles shifts from 7.2 for the original zinc enzyme to 6.6 for the cobalt enzyme . This change suggests that the zinc atom is essential for the catalytic activity of the enzyme similarly to the zinc atom in carboxypeptidase A.

J Bacteriol, 1996 Oct, 178(19), 5719 - 31
Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock; Tsui HC et al.; The amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of Escherichia coli contains genes that are important for diverse cellular functions, including DNA mismatch repair (mutL), tRNA modification (miaA), pleiotropic regulation (hfq), and proteolysis (hflX-hflK-hflC) . We show that this superoperon contains three E simga(32)-dependent heat shock promoters, P(mutL)HS,P(miaA)HS, and P1(hfq)HS, in addition to four E sigma(70)-dependent promoters, P(mutL), P(miaA), P2(hfq), and P3(hfq) . Transcripts from P(mutL)HS and P(miaA)HS were most prominent in vivo during extreme heat shock (50 degrees C), whereas P1(hfq)HS transcripts were detectable under nonshock conditions and increased significantly after heat shock at 50 degrees C . The P(mutL)HS, P(miaA)HS, and P1(hfq)HS transcripts were not detected in an rpoH null mutant . All three promoters were transcribed by E sigma (32) in vitro at 37 degrees C and contain -35 and -10 regions that resemble the E sigma(32) consensus . In experiments to assess the possible physiological relevance of the P(mutL)HS and P(miaA)HS promoters, we found that E . coli prototrophic strain MG 1655 increased in cell mass and remained nearly 100% viable for several hours at 50 degrees C in enriched media . In these cells, a significant fraction of mutL and hfq-hflA region transcripts were from P(mutL)HS and P1(hfq)HS, respectively, and the amounts of the miaA, hfq, hflX, hflK, and hflC transcripts increased in comparison with those in nonstressed cells . The cellular amounts of MutL and the hfq gene product (HF-I protein) were maintained during heat shock at 44 or 50 degrees C . Consistent with their expression patterns, miaA and hfq were essential for growth and viability, respectively, at temperatures of 45 degrees C and above . Together, these results suggest that there is a class of E sigma(32) promoters that functions mainly at high temperatures to ensure E . coli function and survival.

J Bacteriol, 1996 Oct, 178(19), 5573 - 8
Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis; Douglas AL et al.; On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6 . The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations . The 5' extent of P2 was located at bp -42 . Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription . Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity . Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength . Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity . Unlike the partially purified C . trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter . Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E . coli holoenzyme in vitro, suggesting that C . trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E . coli sigma70-holoenzyme.

Curr Microbiol, 1996 Oct, 33(4), 270 - 4
Cooperative interaction between Cra and Fnr in the regulation of the cydAB operon of Escherichia coli; Ramseier TM et al.; In vivo and in vitro experiments are reported demonstrating that the catabolite repressor-activator (Cra) protein (formerly designated FruR) regulates expression of the cydAB operon of Escherichia coli encoding cytochrome d oxidase . The Fnr protein is required for Cra-mediated transcriptional control, but the ArcA protein antagonizes the response to Cra . The results establish that Fnr, ArcA, and Cra exert their effects in an interdependent fashion.

Radiology, 1996 Oct, 201(1), 199 - 205
Experimental pyelonephritis in piglets: diagnosis with MR imaging; Pennington DJ et al.; PURPOSE: To compare findings at magnetic resonance (MR) imaging with those at histopathologic examination in the detection of experimentally induced pyelonephritis in piglets . MATERIALS AND METHODS: MR imaging was performed in 23 piglets with and nine piglets without experimentally induced pyelonephritis . Escherichia coli were injected into the bladder of the 23 piglets with surgically created vesicoureteral reflux . Imaging was performed with unenhanced and contrast material-enhanced T1-weighted and fast multiplanar inversion-recovery (IR) and fast spinecho T2-weighted sequences . MR images and pathologic findings were reviewed independently by two pediatric radiologists and a pathologist, respectively, in a blinded fashion . RESULTS: Sixty-four kidneys and 192 renal zones were evaluated . Coronal gadolinium-enhanced fast multiplanar IR imaging was the only sequence that was sensitive and specific for the diagnosis of pyelonephritis . For the two reviewers, respectively, sensitivity was 85% (n = 75) and 92% (n = 81) of 88 histopathologically positive zones and specificity was 95% (n = 99) and 94% (n = 98) of 104 pathologically negative zones . Findings at gadolinium-enhanced fast multiplanar IR imaging were not statistically different from findings at histopathologic examination in the detection of pyelonephritis . Interobserver reproducibility for the contrast-enhanced fast multiplanar IR sequence was excellent (kappa statistic = 0.82 and 0.90, respectively, for interpretation of a renal zone and of a kidney) . CONCLUSION: Contrast-enhanced fast multiplanar IR imaging is a sensitive and specific test for detection of experimental pyelonephritis in this piglet model.

Mol Cell Biol, 1996 Oct, 16(10), 5477 - 90
Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein; Weng Y et al.; mRNA degradation is an important control point in the regulation of gene expression and has been linked to the process of translation . One clear example of this linkage is the nonsense-mediated mRNA decay pathway, in which nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene . For the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway . Biochemical analysis of the wild-type Upf1p demonstrates that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities . A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs, leading to the production of enough functional product to overcome an auxotrophy resulting from a nonsense mutation . A genetic and biochemical study of the UPF1 gene was undertaken in order to understand the mechanism of Upf1p function in the nonsense-mediated mRNA decay pathway . Our analysis suggests that Upf1p is a multifunctional protein with separable activities that can affect mRNA turnover and nonsense suppression . Mutations in the conserved helicase motifs of Upf1p that inactivate its mRNA decay function while not allowing suppression of leu2-2 and tyr7-1 nonsense alleles have been identified . In particular, one mutation located in the ATP binding and hydrolysis motif of Upf1p that changed the aspartic and glutamic acid residues to alanine residues (DE572AA) lacked ATPase and helicase activities, and the mutant formed a Upf1p:RNA complex in the absence of ATP; surprisingly, however, the Upf1p:RNA complex dissociated as a consequence of ATP binding . This result suggests that ATP binding, independent of its hydrolysis, can modulate Upf1p:RNA complex formation for this mutant protein . The role of the RNA binding activity of Upf1p in modulating nonsense suppression is discussed.

Mol Cell Biol, 1996 Oct, 16(10), 5458 - 65
A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains; Zamir I et al.; Ligand-independent transcriptional repression is an important function of nuclear hormone receptors . An interaction screen with the repression domain of the orphan receptor RevErb identified N-CoR, the corepressor for thyroid hormone receptor (TR) and retinoic acid receptor (RAR) . N-CoR is likely to be a bona fide transcriptional corepressor for RevErb because (i) RevErb interacts with endogenous N-CoR, (ii) ectopic N-CoR potentiates RevErb-mediated repression, and (iii) transcriptional repression by RevErb correlates with its ability to bind N-CoR . Remarkably, a region homologous to the CoR box which is necessary for TR and RAR to interact with N-CoR is not required for RevErb . Rather, two short regions of RevErb separated by approximately 200 amino acids are required for interaction with N-CoR . The primary amino acid sequence of the N-terminal region of RevErb essential for N-CoR interaction is not homologous to that of TR or RAR, whereas similarities exist among the C-terminal domains of the receptors . N-CoR contains two adjacent but distinct interaction domains, one of which binds tightly to both RevErb and TR whereas the other binds more weakly and differentially interacts with the nuclear receptors . These results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR.

Mol Cell Biol, 1996 Oct, 16(10), 5419 - 26
Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III; Bai L et al.; The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription . We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits . Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies . Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta . Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro . In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations . Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF.

J Immunol, 1996 Oct 1, 157(7), 2989 - 97
Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen; Rheinnecker M et al.; We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites . We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4{Fuc alpha 1-->3} GlcNAc beta 1-->3R) . For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created . The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w . of a dimer and tetramer, respectively . Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment . In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

Am Surg, 1996 Oct, 62(10), 793 - 9
Small intestinal absorption during endotoxemia in swine; Kanno S et al.; We studied the effects of systemic endotoxemia on small intestinal absorption in an in vivo animal model . Seven adolescent Yorkshire swine underwent creation of 25 cm distal ileal Thiry-Vella fistulae . After 1 week recovery, the fistulae were perfused with a solution of glucose and electrolytes labeled with 14C-PEG, and net absorption of water, Na+, Cl-, and glucose was calculated . Animals were studied under three different conditions: (1) Basal fasting state, (2) immediately after intravenous injection of E . coli lipopolysaccharide (LPS; 250 micrograms/kg), and (3) 24 hours after LPS . Water, Na+, and Cl- absorption was significantly reduced 2 hours after LPS, but recovered to baseline values by the third hour after LPS . Twenty-four hours after LPS water, Na+, and Cl- absorption was significantly decreased below baseline values . Glucose absorption after LPS paralleled that of water and electrolytes, except that the transient early recovery was not observed . Histological studies of the ileum after LPS showed marked epithelial inflammation at 6 hours, villous atrophy at 24 hours, and signs of recovery at 7 days . Intestinal absorption of water, electrolytes, and glucose is adversely affected in the immediate and early periods after an endotoxemic episode, but the histological epithelial injury secondary to endotoxemia is reversible.

J Mol Evol, 1996 Oct, 43(4), 357 - 73
Secondary structure and patterns of evolution among mammalian mitochondrial 12S rRNA molecules; Springer MS et al.; Forty-nine complete 12S ribosomal RNA (rRNA) gene sequences from a diverse assortment of mammals (one monotreme, 11 marsupials, 37 placentals), including 11 new sequences, were employed to establish a "core" secondary structure model for mammalian 12S rRNA . Base-pairing interactions were assessed according to the criteria of potential base-pairing as well as evidence for base-pairing in the form of compensatory mutations . In cases where compensatory evidence was not available among mammalian sequences, we evaluated evidence among other vertebrate 12S rRNAs . Our results suggest a core model for secondary structure in mammalian 12S rRNAs with deletions as well as additions to the Gutell (1994: Nucleic Acids Res . 22) models for Bos and Homo . In all, we recognize 40 stems, 34 of which are supported by at least some compensatory evidence within Mammalia . We also investigated the occurrence and conservation in mammalian 12S rRNAs of nucleotide positions that are known to participate in the decoding site in E . coli . Twenty-four nucleotide positions known to participate in the decoding site in E . coli also occur among mammalian 12S rRNAs and 17 are invariant for the same base as in E . coli . Patterns of nucleotide substitution were assessed based on our secondary structure model . Transitions in loops become saturated by approximately 10-20 million years . Transitions in stems, in turn, show partial saturation at 20 million years but divergence continues to increase beyond 100 million years . Transversions accumulate linearly beyond 100 million years in both stems and loops although the rate of accumulation of transversions is three- to fourfold higher in loops . Presumably, this difference results from constraints to maintain pairing in stems.

J Virol, 1996 Oct, 70(10), 7108 - 15
The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels; Ewart GD et al.; Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells . It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum . However, the molecular mechanisms employed by Vpu in performance of these functions are unknown . Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes . In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers . The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions . A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E . coli plasma membrane.

J Virol, 1996 Oct, 70(10), 7030 - 8
Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted; Zhou H et al.; Although adenovirus vectors offer many advantages, it would be desirable to develop vectors with improved expression and decreased toxicity . Toward this objective, an adenovirus vector system with deletion of both the El and E2a regions was developed . A 5.9-kb fragment of the adenovirus type 5 (Ad5) genome containing the E2a gene and its early and late promoters was transfected into 293 cells . A complementing cell line, designated 293-C2, expressed the E2a mRNA and protein and was found to complement the defect in Ad5 viruses with temperature-sensitive or deletion mutations in E2a . A deletion of 1.3 kb removing codons 40 to 471 of the 529 amino acids of E2a was introduced into plasmids for preparation of viruses and vectors . An Ad5 virus with disruption of the El gene and deletion of E2a grew on 293-C2 cells but not on 293 cells . Vectors with E1 and E2a deleted expressing Escherichia coli beta-galactosidase or human alpha1-antitrypsin were prepared and expressed the reporter genes after intravenous injection into mice . This vector system retains sequences in common between the complementing cell line and the vectors, including 3.4 kb upstream and 1.1 kb downstream of the deletion . These vectors have potential advantages of increased capacity for insertion of transgene sequences, elimination of expression of E2a, and possibly reduction in expression of other viral proteins . Although the titers of the vectors with deleted are about 10- to 30-fold below those of vectors with E2a wild-type regions, the former vectors are suitable for detailed studies with animals to evaluate the effects on host immune responses, on duration of expression, and on safety.

J Virol, 1996 Oct, 70(10), 6694 - 700
Activity of purified hepatitis C virus protease NS3 on peptide substrates; Steinkuhler C et al.; The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction . Experiments were carried out to optimize protease activity . Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength . C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate . Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein . The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A . A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer . From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.

J Virol, 1996 Oct, 70(10), 6658 - 64
Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1; Ho CK et al.; We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA . This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap . Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity . With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus . We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.

Biochim Biophys Acta, 1996 Sep 30, 1276(3), 195 - 202
Structural factors of rotenone required for inhibition of various NADH-ubiquinone oxidoreductases; Ueno H et al.; We performed a structure-activity study of a series of synthetic rotenone analogues to elucidate the structural factors of rotenone required for inhibition and to probe the structural properties of the rotenone binding site of various NADH-ubiquinone oxidoreductases (NDH), including both proton-pumping (NDH-1) and non-proton-pumping (NDH-2) enzymes, from bovine heart mitochondria, potato tuber (Solanum tuberosum L.) mitochondria and Escherichia coli (GR 19N) plasma membranes . Using a benzyloxy group as a substitute for the E-ring moiety of natural rotenone, systematically selected structural modifications of the A-ring became feasible . The inhibitory potency of bovine NDH markedly varied depending upon structural modifications of the A-ring . The native chemical structure (2,3-dimethoxy substitution) appeared to be the most favorable for the activity . The spatial location of the hydrogen-bond acceptable methoxy oxygens may be important for tight fitting into the binding site . However, replacing one of the two methoxy groups by an ethoxy group almost completely retained the activity, indicating that the binding environment of the A-ring moiety is spacious enough to accommodate a substituent larger than the methoxy group . The manner of action of the derivative lacking the 12-C = O group in the C-ring differed from that of natural rotenone, indicating that this functional group is important for supporting the inhibitory action of natural rotenone itself . Regarding potato tube and E . coli NDH-1, the sensitivity of the two enzymes to the inhibition by rotenone analogues was much lower than that of the bovine enzyme . The 2,3-dimethoxy substitution was the most favorable for the activity with potato NDH-1, whereas this substitution pattern was not necessarily the best with E . coli NDH-1 . A rule governing inhibitory potency depending upon structural modifications was ambiguous for the two enzymes because of a small variation in the inhibitory potencies . These findings indicated that the local binding environment of the A-ring moiety of rotenone in bovine NDH is specific and differs considerably from that in potato and E . coli NDH-1.

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Last modified: May 25, 2005