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J Bacteriol, 1992 Aug, 174(16), 5424 - 9
Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding; Rinken R et al.; Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype . In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble . Remarkably, in recD cells about 25% of the DNA was rendered acid soluble . The DNA degradation in recD cells depended on intact recB and recC genes . The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I) . In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant . Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives . The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation . It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants . The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.

Virology, 1992 Aug, 189(2), 803 - 7
Selective inhibition of the 3'-to-5' exonuclease activity associated with Epstein-Barr virus DNA polymerase by ribonucleoside 5'-monophosphates; Tsurumi T; Epstein-Barr virus (EBV) DNA polymerase possesses a proofreading 3'-to-5' exonuclease activity (Tsurumi, T . (1991) Virology 182, 376-381) . The 3'-to-5' exonuclease activity can be selectively inhibited by ribonucleoside 5'-monophosphates, while no inhibition of the DNA polymerase activity can be observed even when the template/primer concentrations are rate-limiting . Deoxynucleoside monophosphates except 5'dGMP have almost no effect on the exonuclease activity . Of the four ribonucleoside monophosphates, 5'GMP is the most potent (62% inhibition at 5 mM) . The kinetic study shows that 5'-GMP inhibits the exonuclease activity competitively with respect to DNA template/primer . During DNA polymerization process the EBV DNA polymerase catalyzes the DNA-dependent conversion of complementary deoxynucleoside triphosphate to monophosphate form . With poly(dT).oligo(rA) as a template primer, selective inhibition of the exonuclease activity by 5'-GMP results in a decrease in the amount of free dAMP generated which is complementary to the template DNA, suggesting the functional relationship between the editing exonuclease activity and the chain elongation activity of the EBV DNA polymerase molecule.

Virology, 1992 Aug, 189(2), 629 - 39
An in vivo study of a glycoprotein gIII-negative bovine herpesvirus 1 (BHV-1) mutant expressing beta-galactosidase: evaluation of the role of gIII in virus infectivity and its use as a vector for mucosal immunization; Liang X et al.; We constructed a recombinant BHV-1 in which the glycoprotein gIII gene was replaced by the Escherichia coli lacZ gene . The resultant virus mimics the simple gIII deletion mutant in its growth characteristics in cell culture; however, it expresses beta-galactosidase in virus-infected cells . Further characterization of its virulence and the immune responses elicited by it was conducted in cattle . The mutant virus retained the ability to establish an infection when administered intranasally . Infected animals were also capable of transmitting virus to sentinel penmates . However, the mutant virus showed a reduced replication efficiency in the respiratory tract of cattle, as manifested by significantly lower virus shedding and a shorter duration of shedding when compared to wild-type (wt) BHV-1 infections . The mutant virus induced an efficient anti-BHV-1 antibody response and convalescent cattle were fully protected from subsequent wt virus challenge . In addition, cattle infected with the lacZ-expressing virus developed antibodies to beta-galactosidase . Our results demonstrate that the presence of gIII is not a prerequisite for BHV-1 infection; however, gIII does play an important role in maintaining virus replication efficacy in its natural host . With respect to developing BHV-1 as a vaccine vector, our results indicate that deletion of the gIII gene, which partially attenuates the virus and serves as a vaccine virus marker, does not compromise immunogenicity to BHV-1 . Most importantly, this vector is effective in delivering foreign antigens to mucosal surfaces of the respiratory tract.

Virology, 1992 Aug, 189(2), 568 - 82
Temperature-sensitive polioviruses containing mutations in RNA polymerase; Burns CC et al.; Site-directed mutagenesis was performed to change the wild-type residue (asparagine) to aspartate, histidine, or tyrosine at amino acid 424 of the poliovirus RNA polymerase, 3Dpol . The mutations were introduced into plasmids containing full-length viral cDNA and plasmids which direct the expression of 3Dpol in Escherichia coli . Mutant viruses, recovered after transfection of HeLa cells with RNA transcripts of the full-length clones, produced small plaques at 32 degrees . In addition, the plaquing efficiency was decreased for all three mutants at 37 degrees, compared to 32 degrees . The polyprotein processing of all mutant viruses was normal at the temperatures tested, suggesting that the mutant plaque phenotypes were not due to incorrect processing of viral proteins . Analyses of viral RNA synthesis in infected cells and of the polymerase activities of mutant enzymes produced in E . coli suggested the following: (1) The his424 mutant enzyme appeared to be defective in the initiation of plus-strand RNA synthesis in HeLa cells . (2) The asp424 mutant enzyme appeared unable to assume proper conformation for active polymerase function when synthesized at 37 degrees . (3) The tyr424 mutant enzyme was totally inactive when synthesized in E . coli at 37 degrees.

Exp Cell Res, 1992 Aug, 201(2), 462 - 9
Ultraviolet mutagenesis in human lymphocytes: the effect of cellular transformation; Parris CN et al.; We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells . To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189 . Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058) . pZ189 was treated with uv and introduced into the four cell lines by electroporation . Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli . Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines . Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%) . In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells . Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared . Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.

J Virol, 1992 Aug, 66(8), 4884 - 92
The UL8 subunit of the herpes simplex virus helicase-primase complex is required for efficient primer utilization; Sherman G et al.; The herpes simplex virus (HSV) type 1 helicase-primase is a three-protein complex, consisting of a 1:1:1 association of UL5, UL8, and UL52 gene products (J.J . Crute, T . Tsurumi, L . Zhu, S . K . Weller, P . D . Olivo, M . D . Challberg, E . S . Mocarski, and I . R . Lehman, Proc . Natl . Acad . Sci . USA 86:2186-2189, 1989) . We have purified this complex, as well as a subcomplex consisting of UL5 and UL52 proteins, from insect cells infected with baculovirus recombinants expressing the appropriate gene products . In confirmation of previous reports, we find that whereas UL5 alone has greatly reduced DNA-dependent ATPase activity, the UL5/UL52 subcomplex retains the activities characteristic of the heterotrimer: DNA-dependent ATPase activity, DNA helicase activity, and the ability to prime DNA synthesis on a poly(dT) template . We also found that the primers made by the subcomplex are equal in length to those synthesized by the UL5/UL8/UL52 complex . In an effort to uncover a role for UL8 in HSV DNA replication, we have developed a model system for lagging-strand synthesis in which the primase activity of the helicase-primase complex is coupled to the activity of the HSV DNA polymerase on ICP8-coated single-stranded M13 DNA . Using this assay, we found that the UL8 subunit of the helicase-primase is critical for the efficient utilization of primers; in the absence of UL8, we detected essentially no elongation of primers despite the fact that the rate of primer synthesis on the same template is undiminished . Reconstitution of lagging-strand synthesis in the presence of UL5/UL52 was achieved by the addition of partially purified UL8 . Essentially identical results were obtained when Escherichia coli DNA polymerase I was substituted for the HSV polymerase/UL42 complex . On the basis of these findings, we propose that UL8 acts to increase the efficiency of primer utilization by stabilizing the association between nascent oligoribonucleotide primers and template DNA.

J Virol, 1992 Aug, 66(8), 4803 - 13
Transcriptional synergy by the Epstein-Barr virus transactivator ZEBRA; Carey M et al.; ZEBRA is an Epstein-Barr virus (EBV) transcriptional activator that mediates a genetic switch between the latent and lytic states of the virus by binding to the promoters of genes involved in lytic DNA replication and activating their transcription . A computer survey revealed that 9 of 23 potential or known ZEBRA-responsive EBV genes contained two or more upstream binding sites; this suggested that ZEBRA can stimulate transcription synergistically . By using a series of synthetic promoters bearing one, two, three, five, and seven upstream recognition sites, we showed that ZEBRA activates transcription synergistically when templates bearing multiple sites were compared with a template bearing a single site . This phenomenon was observed in both uninfected and EBV-infected B-lymphoid cells and in vitro in a HeLa cell nuclear extract . DNase I footprinting was used to show that the synergy was not due to cooperative DNA binding mediated by direct contact between ZEBRA dimers . The in vitro experiments revealed two manifestations of synergy . One was seen when the levels of transcription observed with the same amounts of ZEBRA added to templates bearing different numbers of sites were compared . The other was observed when the two lowest concentrations of ZEBRA that stimulated measurable transcription from any given template were compared . On the basis of both the number of sites and the calculated Kd of ZEBRA for a single site, we estimated that the critical concentration of ZEBRA needed to elicit transcriptional synergy corresponds to a site occupancy of two or three bound ZEBRA dimers . Our results have biologic implications for both the EBV lytic cycle and other processes in which the concentration of an activator changes either temporally or spatially.

J Bacteriol, 1992 Aug, 174(15), 5168 - 70
Cloning and sequencing of Escherichia coli mutR shows its identity to topB, encoding topoisomerase III; Schofield MA et al.; We have cloned and sequenced the mutR gene from Escherichia coli, which results in an increased frequency of spontaneous deletions, by using a strain carrying a Tn10 derivative inserted into mutR . The analysis of 1,286 bp of mutR sequence shows that this gene is identical to the topB gene, which encodes topoisomerase III . The increased deletion formation is the first reported phenotype for cells lacking topoisomerase III, and this suggests that topoisomerase III is involved in reactions that normally reduce the levels of spontaneous deletions.

J Bacteriol, 1992 Aug, 174(15), 5079 - 85
IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions; Belogurov AA et al.; The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function . ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation . The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli . Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity . Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E . coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB . However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI . The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation . We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively . Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.

J Bacteriol, 1992 Aug, 174(15), 4907 - 12
Mutations in a central highly conserved non-DNA-binding region of OmpR, an Escherichia coli transcriptional activator, influence its DNA-binding ability; Brissette RE et al.; OmpR is a transcriptional activator for the expression of outer membrane porin genes ompF and ompC in Escherichia coli . Its C-terminal half has been identified as the DNA-binding domain (K . Tsung, R . Brissette, and M . Inouye, J . Biol . Chem . 264:10104-10109, 1989) . Recent studies have indicated that the N-terminal non-DNA-binding domain of OmpR is involved in modulating OmpR function through interaction with the EnvZ protein, a kinase and phosphatase for OmpR . We isolated and characterized two mutations, G94D and E111K, in the N-terminal domain of OmpR and one mutation, R182C, in the DNA-binding domain of OmpR . All three mutations abolished the ability of OmpR to bind to the ompF and ompC promoters in vivo, thus giving an OmpF- OmpC- phenotype . The decreased DNA-binding ability of the mutant OmpRs was not due to diminished phosphorylation of their N termini, since all the mutant OmpRs were found to be normally phosphorylated by EnvZ in vitro . The mutant OmpRs produced from multicopy plasmids were also found to inhibit completely the production of OmpF and OmpC in wild-type cells, and the complete inhibition depended on the function of EnvZ which was produced in cis or in trans from plasmids . The relationship of the possible alterations in OmpR by the mutations with the observed diminished binding ability is discussed.

Protein Sci, 1992 Aug, 1(8), 1007 - 13
Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates; Sturzebecher J et al.; BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process . Until now, no X-ray and NMR structures of BM 06.022 were available . Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA . Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties . This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.

Nat Genet, 1992 Aug, 1(5), 372 - 8
Adenovirus-mediated in vivo gene transfer and expression in normal rat liver; Jaffe HA et al.; Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes . In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human alpha 1-antitrypsin cDNA (Ad-alpha 1AT) synthesized and secreted human alpha 1AT for 4 weeks . In rats, in vivo intraportal administration of a recombinant Ad containing the E . coli lacZ gene, was followed by expression of beta-galactosidase in hepatocytes 3 days after infection . Intraportal infusion of Ad-alpha 1AT produced detectable serum levels of human alpha 1AT for 4 weeks . Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders.

Cell Biophys, 1992 Aug-Dec, 21(1-3), 1 - 12
Expression of a Chlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein; Malinowski DP et al.; A single-chain antibody fragment has been constructed for an antibody that binds to the Chlamydia specific carbohydrate structure of the lipopolysaccharide . Single-chain protein was expressed and secreted into the periplasmic space of E . coli as a fusion protein with the maltose binding protein . The fusion protein was purified in one step by virtue of its ability to bind to maltose . In a sandwich ELISA, the eluted protein bound Chlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein . The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.

Acta Virol, 1992 Aug, 36(4), 337 - 46
Bacterially expressed core and envelope proteins of human immunodeficiency virus type-1 (HIV-1): comparative evaluation in detection of type-specific antibodies; Kovac J et al.; Recombinant proteins derived from immunodominant conserved domains of HIV-1 env and gag genes were synthesized in E . coli . An immunoblot system using total cell lysates was employed for the analysis of recombinant bacterial clones . Together 427 serum samples obtained from asymptomatic anti-HIV seropositive individuals, AIDS patients, healthy donors and persons suffering from various conditions were comparatively evaluated for the presence of HIV-1 antibodies using recombinant peptides and commercially available western blot (WB) and ELISA assays . The recombinant antigen product of plasmid pEX41 was found to be superior, with respect to sensitivity and specificity, to the viral gp41 which represents a diagnostically important constituent of the WB.

Biochim Biophys Acta, 1992 Jul 31, 1122(2), 212 - 8
Identification of the histidine residue in Escherichia coli isocitrate lyase that reacts with diethylpyrocarbonate; Rua J et al.; Escherichia coli isocitrate lyase was inactivated by diethylpyrocarbonate in a pseudo-first-order process . The enzyme was completely inactivated by modification of a single histidine residue, but slower modification of further residues also occurred . The substrate, isocitrate, and products, glyoxylate and succinate, protected against inactivation by diethylpyrocarbonate but this was not simply due to binding at the active site . Treatment of the inactivated enzyme with hydroxylamine led to only partial recovery of activity . Diethylpyrocarbonate also reacted with sulphydryl groups in isocitrate lyase, as judged by titrations with Nbs2, but this reaction was not responsible for the failure of hydroxylamine to reactivate the enzyme fully . The reactivity of isocitrate lyase to diethylpyrocarbonate declined with pH, following a titration curve for a group of pKa 6.1 . Isolation and sequencing of ethoxyformylated peptides showed that the major site of modification by diethylpyrocarbonate was histidine residue 306.

Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 811 - 8
Signal peptide for peroxisomal targeting: replacement of an essential histidine residue by certain amino acids converts the amino-terminal presequence of peroxisomal 3-ketoacyl-CoA thiolase to a mitochondrial signal peptide; Osumi T et al.; The role of the histidine residue at position -17 of the amino-terminal signal peptide of rat peroxisomal 3-ketoacyl-CoA thiolase was studied in vivo, employing site-directed mutagenesis . Among the nine amino acids tested, only glutamine could partially substitute for the histidine . Mutants carrying basic amino acids, arginine and lysine, and hydrophobic residues, leucine and valine, in place of histidine were all translocated to mitochondria, but not to peroxisomes . These results indicate that the signal peptide of the thiolase is recognized by a mechanism totally different from that for the SKL motif, a known peroxisomal targeting signal . Relationship of the thiolase signal peptide to those of mitochondrial proteins is discussed.

Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 686 - 9
Identification of the iron-binding histidine residues in soybean lipoxygenase L-1; Steczko J et al.; Lipoxygenases constitute a class of non-heme, non-sulfur iron dioxygenases acting upon lipids possessing a 1,4-cis-cis-pentadiene moiety . The iron is known to be essential for activity . A motif of six histidine residues has been found in all of the thirteen lipoxygenases, from both plant and animal sources, whose sequences have been reported . We had previously obtained mutant proteins in which each of the 6 conserved histidines of soybean lipoxygenase L-1 had been replaced and found that the mutants H499Q, H504Q (or H504S) and H690Q had no detectable enzymatic activity . We have now found that these inactive proteins contain no Fe, although they have the same electrophoretic mobility as wild-type L-1 under both denaturing and non-denaturing conditions and react with anti-L-1 antibodies.

Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 1081 - 6
Phosphorylation of the recombinant spliced variants of the alpha-sub-unit of the stimulatory guanine-nucleotide binding regulatory protein (Gs) by the catalytic sub-unit of protein kinase A; Pyne NJ et al.; Both GS alpha-1 and GS alpha-4 were phosphorylated by the purified catalytic sub-unit of protein kinase A . Phosphate incorporation into 220 pmol and 190 pmol of GS alpha-4 and GS alpha-1 after a 1 hour incubation with kinase was 14 pmol and 10 pmol, respectively . These low levels of phosphorylation are due to the thermal lability of purified recombinant GS alpha . However, the phosphorylation was inhibited by guanine nucleotides (GDP-beta-S, GppNHp and GTP) and is, therefore, a specific event . We suggest that, as for GS alpha phosphorylation by protein kinase C (Pyne et al., 1992), the guanine nucleotide-free form of GS alpha is the most likely substrate . Guanine-nucleotides reduce the lifetime and, therefore availability for phosphorylation, of guanine-nucleotide free GS alpha . GS alpha phosphorylation by protein kinase A in vitro provides preliminary evidence that a similar phosphorylation of GS alpha may be an important regulatory event in cells.

Biochemistry, 1992 Jul 28, 31(29), 6683 - 91
Unfolding of trp repressor studied using fluorescence spectroscopic techniques; Fernando T et al.; The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches . Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor . These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol . The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition . The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews {(1990) Biochemistry 29, 7011} . This indicates that the intensity increase has both dynamic and static origins . To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe . These experiments revealed a protein concentration dependent transition at low urea concentrations . This transition was promoted by tryptophan binding . We ascribe this transition to urea-induced dissociation of repressor tetramers . The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1992 Jul 28, 307(2), 185 - 9
Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin; Rodriguez-Arango E et al.; The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library . It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids . The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus . Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol . wt . 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein . The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor . Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose . The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes . Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.

FEBS Lett, 1992 Jul 28, 307(2), 229 - 32
Serine residues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; Putative transmembrane helix 3 of the tetracycline/H+ antiporter encoded by a transposon, Tn10, contains four serine residues, Ser-77, Ser-82, Ser-91 and Ser-92 . Each of these serine residues was replaced by site-directed mutagenesis . Of these four serine residues, Ser-77 was important for the transport function, and a bulky side chain at position 91 hindered substrate translocation, whereas Ser-82 and Ser-92 did not play any role . Ser-77 and Ser-91 are on the same vertical stripe, that includes the essential Asp-84, on the hydrophilic side of putative helix 3 . These observations suggest that helix 3 is part of the tetracycline translocation channel across the membrane.

J Biol Chem, 1992 Jul 15, 267(20), 14443 - 50
A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate; Carver TE et al.; Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix notation) to examine the effects of distal pocket size on the rates of ligand binding and autooxidation . Leu29 was replaced with Ala, Val, and Phe using the synthetic gene and Escherichia coli expression system of Springer and Sligar (Springer, B . A., and Sligar, S . G . (1987) Proc . Natl . Acad . Sci . U . S . A . 84, 8961-8965) . Structures of the ferric forms of Val29 and Phe29, and the oxy form of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography . The ferric mutant proteins are remarkably isomorphous with the wild type protein except in the immediate vicinity of residue 29 . Thus, the protein structure in the distal pocket of myoglobin can accommodate either a large "hole" (i.e . Ala or Val) or a large side chain (i.e . Phe) at position 29 without perturbation of tertiary structure . Phe29 oxymyoglobin is also identical to the native oxy protein in terms of overall structure and interactions between the bound O2 and His64, Val68, Phe43, and Ile107 . The distance between the nearest side chain atom of residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the Phe29 protein and 4.9 A in native myoglobin . The equilibrium constants for O2 binding to Ala29, Val29, and Leu29 (native) myoglobin are the same, approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29 protein is markedly greater, 15 x 10(6) M-1 . This increase in affinity is due primarily to a 10-fold decrease in the O2 dissociation rate constant for the Phe29 mutant and appears to be the result of stabilizing interactions between the negative portion of the bound O2 dipole and the partially positive edge of the phenyl ring . Increasing the size of residue 29 causes large decreases in the rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37 degrees C . Thus, the Leu29----Phe mutation produces a reduced protein that is remarkably stable and is expressed in E . coli as 100% MbO2 . The selective pressure to conserve Leu29 at the B10 position probably represents a compromise between reducing the rate of autooxidation and maintaining a large enough O2 dissociation rate constant to allow rapid oxygen release during respiration.

J Biol Chem, 1992 Jul 15, 267(20), 14227 - 32
Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease; Chattopadhyay D et al.; Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment . The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass . Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC . The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays . Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440 . Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3693 - 700
Expression and characterisation of the korB gene product from the Streptomyces lividans plasmid pIJ101 in Escherichia coli and determination of its binding site on the korB and kilB promoters; Zaman S et al.; A 0.5kb Spel-BclI fragment containing the pIJ101 korB ORF was cloned into pUC8 under the control of the lacZ promoter, creating pQR206 . In vitro coupled transcription-translation of pQR206 identified a protein product of approximately 10kDa, which corresponds to the predicted molecular weight deduced from the korB sequence . pQR206 was used to express the 10kDa KorB protein in vivo in E . coli . Crude E . coli protein extracts containing KorB were shown to bind to a 0.8kb kilB fragment and a 0.5kb korB fragment in gel retardation assays . DNasel footprinting indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kilB promoter and a 36bp region encompassing the korB promoter.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3651 - 7
The application of Markov chain analysis to oligonucleotide frequency prediction and physical mapping of Drosophila melanogaster; Cuticchia AJ et al.; Here we compare several methods for predicting oligonucleotide frequencies in 691 kb of Drosophila melanogaster DNA . As in previous work on Escherichia coli and Saccharomyces cerevisiae, a relatively simple equation based on tetranucleotide frequencies can be used in predicting frequencies of higher order oligonucleotides . For example, the mean of observed/expected abundances of 4,096 hexamers was 1.07 with a sample standard deviation of .55 . This simple predictor arises by considering each base on the sense strand of D . melanogaster to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate than the random predictor . This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in selecting probes to fingerprint clones in contig mapping . Once again, this equation well predicts the occurrence of higher order oligonucleotides, supporting our hypothesis that this predictor holds in evolutionarily diverse organisms . When ranked from highest to lowest abundance, the observed frequencies of oligomers of a given length are closely tracked by the predicted abundances of a 3rd order Markov chain . Through use of the dependence of oligomer frequencies on base composition, we report a list of oligomers that will be useful for the completion of a cosmid physical map of D . melanogaster . Presently, the library is such that it will be possible to construct large contigs using only 30 oligonucleotide probes to fingerprint cosmids.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3631 - 7
A comparison of several similarity indices used in the classification of protein sequences: a multivariate analysis; Landes C et al.; The present work describes an attempt to identify reliable criteria which could be used as distance indices between protein sequences . Seven different criteria have been tested: i and ii) the scores of the alignments as given by the BESTFIT and the FASTA programs; iii) the ratio parameter, i.e . the BESTFIT score divided by the length of the aligned peptides; iv and v) the statistical significance (Z-scores) of the scores calculated by BESTFIT and FASTA, as obtained by comparison with shuffled sequences; vi) the Z-scores provided by the program RELATE which performs a segment-by-segment comparison of 2 sequences, and vii) an original distance index calculated by the program DOCMA from all the pairwise dotplots between the sequences . These 7 criteria have been tested against the aminoacid sequences of 39 globins and those of the 20 aminoacyl-tRNA synthetases from E . coli . The distances between the sequences were analyzed by the multivariate analysis techniques . The results show that the distances calculated from the scores of the pairwise alignments are not adequately sensitive . The Z-score from RELATE is not selective enough and too demanding in computer time . Three criteria gave a classification consistent with the known similarities between the sequences in the sets, namely the Z-scores from BESTFIT and FASTA and the multiple dotplot comparison distance index from DOCMA.

J Biol Chem, 1992 Jul 25, 267(21), 14799 - 803
Multiple forms of ubiquitin-activating enzyme E1 from wheat . Identification of an essential cysteine by in vitro mutagenesis; Hatfield PM et al.; Ubiquitin-activating enzyme, E1, directs the ATP-dependent formation of a thiol ester linkage between itself and ubiquitin . The energy in this bond is ultimately used to attach ubiquitin to various intracellular proteins . We previously reported the isolation of multiple E1s from wheat and the characterization of a cDNA encoding this protein (UBA1) . We now report the derived amino acid sequence of two additional members of this gene family (UBA2 and UBA3) . Whereas the amino acid sequence of UBA2 is nearly identical to UBA1, the sequence of UBA3 is significantly different . Nevertheless, the protein encoded by UBA3 catalyzes the ATP-dependent activation of ubiquitin in vitro . Comparison of derived amino acid sequences of genes encoding E1 from plant, yeast, and animal tissues revealed 5 conserved cysteine residues, with one potentially involved in thiol ester bond formation . To identify this essential residue, codons corresponding to each of the 5 cysteines in UBA1 were individually altered using site-directed mutagenesis . The mutagenized enzymes were expressed in Escherichia coli and assayed for their ability to activate ubiquitin . Only substitution of the cysteine at position 626 abolishes E1 activity, suggesting that this residue forms the thiol ester linkage with ubiquitin.

J Biol Chem, 1992 Jul 25, 267(21), 14611 - 5
Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein; Kikuchi A et al.; smg GDS and rho GDI are stimulatory and inhibitory GDP/GTP exchange proteins, respectively, for a group of ras p21-related small GTP-binding proteins (G proteins) . rho p21 is a common substrate small G protein for both GDP/GTP exchange proteins . We examined here the functional interactions of these GDP/GTP exchange proteins with rho p21 as a substrate . smg GDS and rho GDI interacted with the GDP-bound form of rho p21 and thereby stimulated and inhibited, respectively, the dissociation of GDP . The inhibitory effect of rho GDI was much stronger than the stimulatory effect of smg GDS . The GDP-bound form of rho p21 formed a complex with rho GDI but not with smg GDS in their simultaneous presence . Since the content of smg GDS was generally less than that of rho GDI in cells, these results suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the smg GDS action during the conversion of rhoA p21 from the GDP-bound inactive form to the GTP-bound active form in intact cells . On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21 . This ADP-ribosylation was inhibited by rho GDI much more efficiently than by smg GDS . These results suggest that rho GDI may mask the putative effector region of rho p21 and thereby inhibit its interaction with the target protein even in the presence of smg GDS . Thus, both smg GDS and rho GDI are important to regulate the rho p21 activity and action in cooperation with each other.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3645 - 50
RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins; Harper DS et al.; We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins . Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins . However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B" . Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA . Furthermore, D25 bound U1 RNA when transfected into mammalian cells . Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".

J Biol Chem, 1992 Jul 25, 267(21), 15184 - 92
Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding; Ulbrandt ND et al.; SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells . Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids . In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined . The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids . These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer . It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein . This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis . These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide . This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol . The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.

J Biol Chem, 1992 Jul 25, 267(21), 14713 - 20
DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein; Gunasekera A et al.; The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3' . Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved . In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10 . All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP . Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group . Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group . dam methylation has little effect on CAP.DNA complex formation . The thermodynamically defined consensus DNA site spans 28 base pairs . All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center . The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.

J Biol Chem, 1992 Jul 25, 267(21), 14559 - 62
pH dependence of proton translocation by Escherichia coli; Verkhovskaya M et al.; Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase . Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8 . At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection . The pH effect was confined to cytochrome o-linked activity . It was not present when cytochrome d generated the electrochemical proton gradient . This makes it improbable that the Na+/H+ antiporter is responsible . The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.

Biochem Pharmacol, 1992 Jul 22, 44(2), 199 - 204
Substrate specificity of Escherichia coli thymidine phosphorylase for pyrimidine nucleosides with anti-human immunodeficiency virus activity; Schinazi RF et al.; Various nucleoside antiviral agents and their metabolites were examined for their ability to be cleaved across the glycosidic bond by Escherichia coli thymidine phosphorylase . The increasing order of susceptibility to cleavage was U greater than T much greater than C derivatives . Nucleosides that were unsaturated in the sugar moiety were more susceptible than saturated ones . 3'-Deoxy-2',3'-didehydrothymidine was a substrate, whereas 3'-azido-, 3'-fluoro-, 3'-oxo- and 3'-thiapyrimidine nucleosides were resistant to this enzyme.

Biochemistry, 1992 Jul 21, 31(28), 6598 - 602
13C isotope effect studies of Escherichia coli aspartate transcarbamylase in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate; Parmentier LE et al.; 13C isotope effects have been measured for the aspartate transcarbamylase holoenzyme (ATCase) and catalytic subunit catalyzed reactions in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate (PALA) . For holoenzyme-catalyzed reactions in the physiological direction with very low levels of L-aspartate as substrate, or with L-cysteine sulfinate as substrate, or in the reverse direction with carbamyl-L-aspartate and phosphate as substrates, the isotope effect data show a slight dependence on PALA concentration . Under these conditions, PALA first stimulates the rate and then inhibits it at higher concentrations . The observed isotope effect at maximum stimulation by PALA is slightly smaller than in the absence of the analog, but as the PALA concentration is increased to reduce the rate to its original value, the observed isotope effect also increases and approaches the value of the isotope effect determined in the absence of PALA . These data suggest that the kinetic properties of the active enzyme are affected by the number of active sites occupied by PALA, indicating communication between subunits, and a mathematical model is proposed which explains our experimental observations . In contrast to these results with the holoenzyme, isotope effects measured for the reaction catalyzed by the isolated catalytic subunits are not altered in the presence of PALA . Taken together, these data are consistent with the two-state model for the homotropic regulation of ATCase.

Biochemistry, 1992 Jul 21, 31(28), 6577 - 84
13C and 15N isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase; Parmentier LE et al.; 13C and 15N isotope effects have been measured for the aspartate transcarbamylase (ATCase) reaction in an effort to elucidate the chemical mechanism of this highly regulated enzyme . The observed 15(V/K(asp))H2O value for the ATCase holoenzyme at saturing carbamyl phosphate and 12 mM L-aspartate is 1.0045 at pH 7.5, and this value remains unchanged in the presence of 5 mM ATP (activator) or 5 mM CTP (inhibitor) . The fact that the isotope effect is not changed by the allosteric modifiers supports the conclusion that the kinetic properties of the active form of ATCase are not influenced by ATP or CTP . The observed 15(V/K(asp)) values for the catalytic subunit of ATCase are also the same as those determined for the holoenzyme, suggesting that the chemical mechanisms of both enzyme species are the same . Quantitative analysis of 13C and 15N isotope effects in both H2O and D2O has led to the proposal of a chemical model for the ATCase reaction which involves a precatalytic conformational change to form an activated complex that facilitates deprotonation of L-aspartate by an enzyme functional group . Nucleophilic attack on the carbonyl carbon of carbamyl phosphate by the alpha-amino group of L-aspartate results in the formation of a tetrahedral intermediate . An intramolecular proton transfer leads to formation of products N-carbamyl-L-aspartate and inorganic phosphate.

Biochemistry, 1992 Jul 21, 31(28), 6570 - 6
13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase; Parmentier LE et al.; 13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase . For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect . This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate . The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers . These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates and modifiers, and there are no enzyme forms having partial activity . Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random . With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate . The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme.

Biochemistry, 1992 Jul 21, 31(28), 6562 - 9
Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase; Turnbull JL et al.; The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined . The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis . The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate . However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate . The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis . By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated . This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jul 21, 31(28), 6402 - 13
Phospholipase A2 engineering . Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73; Dupureur CM et al.; Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli) . According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99 . Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2 . Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics {Berg, O . G., Yu, B.-Z., Rogers, J., & Jain, M . K . (1991) Biochemistry 30, 7283-7297} . The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values . The results indicated little perturbation in the interfacial binding step (E to E*) but ca . 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat . Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site . Tyr-73 appears to play an important structural role . The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2 . The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility . These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.

Biochemistry, 1992 Jul 21, 31(28), 6447 - 53
Proximity between nucleotide/dinucleotide and metal ion binding sites in DNA-dependent RNA polymerase from Escherichia coli; Tyagi SC et al.; In order to understand translocation in transcription, it is important to develop a continuous functional assay for RNA polymerase (RNAP) activity in vitro . Fluorescent derivatives of ATP, UTP, UpA, and CpA with aminonaphthalene-5-sulfonic acid (AmNS) attached to the nucleotide triphosphates via a gamma-phosphoramidate bond or to the dinucleotide monophosphates via a 5'-secondary amine linkage were synthesized {Tyagi, S.C., & Wu, F.Y.-H . (1987) J . Biol . Chem . 262, 10684-10688} . The fluorescent emission spectra of (5'-AmNS)UpA, (5'-AmNS)CpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP overlap the absorption spectrum of co-substituted RNA polymerase (Co-RNAP) and ensure fluorescence resonance energy transfer (FRET) between the fluorescent analog and Co(II) in Co-RNAP . The binding constants at a single site for (gamma-AmNS)ATP, (gamma-AmNS)UTP, (5'-AmNS)UpA, and (5'-AmNS)CpA were observed to be 7.11, 5.26, 0.52, and 0.61 microM, respectively, in Co-RNAP and 5.70, 3.42, 0.12, and 0.21 microM, respectively, in Zn-RNAP . (8-AmTEMPO)ATP, with the spin probe AmTEMPO attached to the C-8 position at ATP {Tyagi, S.C . (1991) J . Biol . Chem . 266, 17936-17940}, and Mn(3'-OCH3)UTP were synthesized . Mn-(II)-substituted RNA polymerase (Mn-RNAP) is prepared . The single site binding constants for (8-Am-TEMPO)ATP and Mn(3'-OCH3)UTP were 3.58 and 2.35 microM in Zn-RNAP and 5.77 and 3.43 microM in Mn-RNAP, respectively . These results indicate that dinucleotides bind much more tightly than mononucleotides to RNAP and that the binding constants are roughly the same for both Co- and Zn-substituted RNAP.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1992 Jul 20, 226(2), 387 - 97
Binding of the arginine repressor of Escherichia coli K12 to its operator sites; Tian G et al.; In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes . In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs . The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine . From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs . The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes . From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes . The bound repressor was shown to induce bending of argF operator DNA . The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes . The main features that distinguish the arginine repressor from other repressors studied in E . coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.

J Mol Biol, 1992 Jul 20, 226(2), 367 - 86
Arginine regulon of Escherichia coli K-12 . A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression; Charlier D et al.; The 12 genes which in E . coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5' non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the repressor, a hexameric protein . In vitro binding experiments with purified repressor (a gift from W . K . Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAb and the bipolar argECBH cluster . A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only . Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves . Symmetrical contacts in the minor groove with A residues have also been identified . Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes . Although the wild-type operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity . Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box . The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect . However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced . Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate Oc type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed . The significance of this lack of correlation is discussed.

FEBS Lett, 1992 Jul 20, 306(2-3), 251 - 6
Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch-clamp; Berrier C et al.; E . coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration-rehydration and studied by patch-clamp . The porins could be closed by voltage pulses under -100 mV . The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl . Rapid fluctuations (in the millisecond range) of about one third (60-70 pS) of the large closure steps were also observed . The data are interpreted as follows: an increase in membrane potential favours the cooperation transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short-lived closed state.

FEBS Lett, 1992 Jul 20, 306(2-3), 103 - 7
A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine; Li N et al.; 1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene . We have isolated, sequenced and expressed a functional tomato (cv Pik-Red) ACC synthase gene in Escherichia coli . ACC synthase expressed in E . coli was inactivated by incubation with S-adenosylmethionine (SAM), the half-time of which was concentration dependent . Mixing the tomato fruit protein extract with the cell-free extract from transformed E . coli did not affect SAM-dependent inactivation of ACC synthase activity . Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E . coli and their structure-function relationships investigated.

Carbohydr Res, 1992 Jul 20, 232(1), 77 - 87
Studies of the interaction of the maltose-binding protein of Escherichia coli, a closed-groove binder, with 4,6-O-ethylidenemalto-oligosaccharides (dp 2-5) and its regioselective labelling with 3-azibutyl 1-thio-alpha-(6-3H)maltoside; Lehmann J et al.; Four malto-oligosaccharides (dp 2-5), each with a 4,6-O-ethylidene group on the glucosyl unit at the non-reducing terminus, were synthesised and used to prove that the maltose-binding protein (MBP) of E . coli is a closed-groove binder . alpha-D-Glucosylation of 3-azibutyl 1-thio-alpha-D-(6-3H)glucopyranoside yielded a 3H-labelled, photolabile 1-thiomaltoside derivative that was used to chemically modify the binding site of MBP . The 3H-labelled peptide containing 83% of the total radioactivity, which was isolated after tryptic cleavage of the modified MBP and sequenced, is part of the closed end of the MBP groove.

Carbohydr Res, 1992 Jul 20, 232(1), 33 - 45
The synthesis and characterisation of 2-O-(6-O-L-glycero-alpha,beta-D-manno-heptopyranosyl-alpha-D-glucopyran osyl)-alpha,beta-D-glucopyranose; Nepogodev SA et al.; The title compounds were synthesised, and appropriate derivatives were characterised by GLC, GLC-MS, and NMR spectroscopy . The GLC and GLC-MS data proved 2-O-(6-O-L-glycero-alpha-D-manno-heptopyranosyl-alpha-D-glucopyranosyl)- D- glucopyranose to be a constituent of the outer-core region of the lipopolysaccharide from Escherichia coli K-12, indicating the heptosyl residue to be linked to the terminal glucopyranose residue.

J Mol Biol, 1992 Jul 20, 226(2), 425 - 32
Electron microscopic study of (A)BC excinuclease . DNA is sharply bent in the UvrB-DNA complex; Shi Q et al.; Nucleotide excision repair in Escherichia coli is initiated by the UvrA, UvrB and UvrC proteins . UvrA is the damage recognition subunit, makes an A2B1 complex with the targeting subunit UvrB, and the complex binds to the lesion site; UvrA dissociates leaving behind a very stable UvrB-DNA complex that is recognized by the trigger subunit, UvrC, and the ensuing UvrB-UvrC heterodimer makes two incisions, one on either side of the lesion . Using electron microscopy, we investigated the structures of these early A, A-B intermediates on DNA containing ultraviolet light photoproducts . UvrA, which is known to bind to DNA as a dimer and produce a DNase I footprint of 33 base-pairs does not change the trajectory of DNA appreciably . The A2B1 complex clearly shows a bipartite structure and its effect on the trajectory of the DNA was not consistently straight or kinked . In contrast, the DNA in the preincision UvrB-DNA complex appears to be severely kinked; 43% of the molecules are bent by 80 degrees or more, with an average bending angle of 127 degrees . It appears that protein-induced bending is an important step on the pathway leading to excision of the damaged nucleotide by (A)BC excinuclease.

J Mol Biol, 1992 Jul 20, 226(2), 399 - 409
Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli; Perreault JP et al.; The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate . The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA . The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+ . An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit . Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro . The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

FEBS Lett, 1992 Jul 20, 306(2-3), 185 - 8
3'-Mercapto-2',3'-dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase; Yuzhakov AA et al.; Four 3'-mercapto-2',3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substrates for calf thymus alpha-DNA polymerase, E . coli DNA polymerase I Klenow fragment, terminal deoxynucleotidyl transferase and reverse transcriptases of AMV, HIV and MLV viruses . It was shown that the analogues selectively and irreversibly terminated DNA chain elongation by AMV and HIV reverse transcriptases and the terminal transferase . Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 537 - 40
Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1; Kolm RH et al.; The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized . The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta . The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis . The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value . Kinetic parameters reflecting binding affinity, S0.5 (substrate concn . giving 1/2V) and I50 (concn . of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme . These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates . The dependence of k(cat.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively . The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 of 7.1 . The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 377 - 81
Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1; Widersten M et al.; Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis . The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn . The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity . Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx . 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger . In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes . Asp99 primarily contributes to catalysis rather than to binding . The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism . The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

Gene, 1992 Jul 15, 116(2), 139 - 50
Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody; Trirawatanapong T et al.; Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host . In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5 . First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides . Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422 . For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR) . The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR . The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397) . A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay . In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.

Eur J Biochem, 1992 Jul 15, 207(2), 733 - 9
A mutation at Gly314 of the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change; Ahmad S et al.; Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity . A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the beta subunit by a Glu residue . The mutant enzyme was partially purified and its trypsin cleavage products examined . The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin . However, the beta subunit of the mutant enzyme retained its ability to bind to NAD-agarose . Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis . All substitutions for Gly314 abolished the activity completely . The enzyme containing the Gly314----Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314----Glu mutation . It is concluded that the mutation in the beta subunit abolished the NADP(H)-induced conformational change in the mutant enzyme . This conformational change, caused by NADP(H) binding, is required to cleave the normal beta subunit at Arg265 by trypsin . The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence {Clarke et al . (1986) Eur . J . Biochem . 158, 647-653}.

Eur J Biochem, 1992 Jul 15, 207(2), 541 - 7
S100P, a novel Ca(2+)-binding protein from human placenta . cDNA cloning, recombinant protein expression and Ca2+ binding properties; Becker T et al.; A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties . Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P . The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield . S100P is a homodimer and has two functional EF hands/polypeptide chain . The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence . The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85) . Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan) . The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity . This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target . In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II . The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.

Eur J Biochem, 1992 Jul 15, 207(2), 521 - 31
Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys; Veitch NC et al.; Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy . Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase . The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form . The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar . This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains . Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances . These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution . The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group . Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study . A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152 . The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.

Eur J Biochem, 1992 Jul 15, 207(2), 441 - 7
Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides; Kuntz DA et al.; Two series of oligopeptides have been synthesized . Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied . Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer . None of these exerted any inhibitory effect . The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system . Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli . The most active peptide, cyclo{-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-}, exerted 50% inhibitory activity at a concentration of 3 microM . The nature of the inhibitory action of one of these compounds cyclo{-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-} was studied in more detail . Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.

Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6535 - 9
Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system; Mankin AS et al.; A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene . The vector construct contains three functional sections: (i) an entire H . halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp . SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli . Transformation of H . halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure . After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost . Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell . Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers . The system has considerable potential for ribosomal engineering.

Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6492 - 6
The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA; Hsieh P et al.; A key step in homologous recombination is the alignment and pairing of homologous DNAs . The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament . We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-{gamma-thio}triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA . RecA can pair as few as eight bases of homology to form such synaptic complexes . The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs . Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.

J Biol Chem, 1992 Jul 15, 267(20), 14328 - 34
Precursor for the light-harvesting chlorophyll a/b-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; Oblong JE et al.; When synthesized in Escherichia coli, the light-harvesting chlorophyll a/b-binding protein (LHCP) precursor accumulates in inclusion-like bodies (Abad, M . S., Oblong, J . E., and Lamppa, G . K . (1991) Plant Physiol . 96, 1220-1227) . In this study we show that after solubilization in 6 M urea and dialysis into 20 mM Tris-HCl (pH 8.0) the recombinant LHCP precursor (preLHCP) was not found as a monomer (31 kDa), but instead produced a heterogeneous population of oligomeric complexes, ranging from 60-300 kDa as determined by gel filtration chromatography . Circular dichroism analysis indicated that the oligomers had folded structure, and that it was composed of both alpha-helix and beta-sheet . Approximately half of recombinant preLHCP found in these complexes was cleavable at the transit peptide-mature protein junction by a soluble chloroplast-processing enzyme in an organelle-free reaction . At 1.5 microM the recombinant precursor inhibited the import of radiolabeled preLHCP and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase generated by reticulocyte lysate translations . When chloroplasts were preincubated with the precursor, followed by their reisolation, import was still blocked, providing evidence that competition between recombinant preLHCP and these substrates occurred at the chloroplast per se . Recombinant preLHCP was visualized on the envelope by immunofluorescence microscopy, and its presence there was mediated by a thermolysin-sensitive factor.

J Biol Chem, 1992 Jul 15, 267(20), 14167 - 74
Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin; Hsu DK et al.; IgE-binding protein (epsilon BP) was originally identified by virtue of its affinity for IgE . It is now known to be a beta-galactoside-binding lectin with the characteristic of an S-type carbohydrate recognition domain . The protein is composed of two domains: the amino-terminal domain consisting of tandem repeats and the carboxyl-terminal domain containing sequences shared by other S-type carbohydrate recognition domains . The amino-terminal domain also contains a number of potential recognition sites for collagenase cleavage . In this study, human epsilon BP was first expressed in Escherichia coli, and the carboxyl-terminal domain (epsilon BP-C) was then generated by collagenase digestion of epsilon BP . By equilibrium dialysis, the association constants of epsilon BP and epsilon BP-C for lactose were found to be similar (6.0 +/- 0.70) x 10(4) M-1 and (4.7 +/- 0.27) x 10(4) M-1, respectively . Both polypeptides contain only one lactose-binding site/molecule . By an assay involving binding of 125I-labeled epsilon BP or epsilon BP-C to solid phase IgE, and inhibition of this binding by saccharides, it was determined that epsilon BP-C retains the saccharide specificity of epsilon BP . Importantly, although unlabeled epsilon BP-C inhibited the binding of the radiolabeled epsilon BP to IgE, unlabeled epsilon BP caused increased binding to IgE, suggesting self-association among epsilon BP molecules . Oligomeric structures resulting from self-association of epsilon BP were confirmed by chemical cross-linking studies . Furthermore, epsilon BP possesses hemagglutination activity on rabbit erythrocytes, whereas epsilon BP-C lacks such activity . Based on these results, we propose a structural model for multivalency of epsilon BP: dimerization or oligomerization of epsilon BP occurs through intermolecular interaction involving the amino-terminal domain.

J Biol Chem, 1992 Jul 15, 267(20), 14122 - 8
Upstream sequence elements required for NarL-mediated activation of transcription from the narGHJI promoter of Escherichia coli; Dong XR et al.; Transcription of the narGHJI operon (encoding nitrate reductase) in Escherichia coli is primarily dependent on the activation of the pleiotropic transacting factor Fnr, which interacts with the promoter through a cis element (Fnr box) located near the transcription start site . Further stimulation of transcription occurs in the presence of nitrate and is dependent on activation of the transacting factor NarL and a cis-acting sequence (NarL box) located approximately 200 base pairs upstream from the transcription start site . To define the structure of the NarL box, alterations in the NarL box region, generated by saturation mutagenesis of the sequence from positions -184 to -202 in the narGHJI promoter of a narG::lacZ fusion-bearing plasmid, were analyzed for their effects on NarL-mediated stimulation of transcription . Single base substitutions that significantly reduced the NarL-mediated stimulation were restricted to a 6-base sequence, TACTCC, located at positions -193 to -198 in the narGHJI promoter . When 2 bases were modified, NarL-mediated stimulation was severely reduced when one or both alterations were located within the 6-base sequence . Attempts to restore NarL-mediated stimulation with an inverted NarL box were not successful . Although previous studies suggested that NarL-mediated stimulation of transcription may occur by a DNA looping mechanism, the results presented here demonstrate that it does not involve the passive formation of a simple DNA loop . Replacement of 94 or 108 bases of the approximately 150 base sequence between the Fnr box and the NarL box with an unrelated sequence resulted in elimination of NarL-mediated stimulation of transcription . Furthermore, shifting of most of the intervening sequence or defined segments of the sequence by 4 bases while maintaining the position of the NarL box relative to sequences required for Fnr-dependent, anaerobic transcription also eliminated the NarL-mediated stimulation . We conclude that in addition to the 6-base NarL box located on a specific face of the promoter DNA, the stimulation of transcription by NarL requires some specific sequences and/or higher order structure specified by the DNA that separates the NarL box from the Fnr box.

J Biol Chem, 1992 Jul 15, 267(20), 14077 - 83
Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins; Santambrogio P et al.; Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains . The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown . Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions . In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains . In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin . The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain . One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.

J Biol Chem, 1992 Jul 15, 267(20), 13986 - 92
cDNA cloning and molecular characterization of MSE55, a novel human serum constituent protein that displays bone marrow stromal/endothelial cell-specific expression; Bahou WF et al.; Hemonectin is a lineage-specific cytoadhesive protein that may be involved in the developmentally regulated adhesion of granulocytic cells to bone marrow stroma . Immunoblot analysis using an anti-hemonectin antibody recognizes two distinct immunoreactive species in endothelial cell lysates (approximately M(r) 65,000) and human serum (approximately M(r) 55,000) . Initial characterization of the 55-kDa protein has now been completed by isolating the cDNA from a human endothelial cell expression library . Sequence analysis of overlapping clones identifies a composite sequence spanning 2030 nucleotides with an open reading frame of 1173 base pairs . No significant sequence similarity was observed on analysis of current GenBank databases . The open reading frame was expressed as a recombinant protein in Escherichia coli and used as an immunogen for the production of a specific polyclonal antibody . Immunoblotting with this antibody identifies a single immunoreactive species of apparent M(r) 55,000 in HUVEC lysates and human serum, confirming that a secreted form normally circulates as a serum constituent protein . This antibody fails to recognize purified hemonectin, suggesting that the M(r) 55,000 protein is not hemonectin . Cross-species Southern blot analysis reveals persistent hybridizing fragments in all species tested, suggestive of a developmentally conserved function . Northern blot analysis demonstrates expression limited to endothelial and bone marrow stromal cells, but not poly(A) RNA from monkey liver, spleen, brain, lung, and kidney . On this basis, we have designated this novel protein MSE55, for marrow stromal/endothelial cell protein with a molecular mass of 55,000 daltons . Its tissue-specific expression may suggest a functional role in hematopoiesis.

J Biol Chem, 1992 Jul 15, 267(20), 13843 - 50
Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation; Chen J et al.; The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A . E., Tesmer, V . M., Manly, S . P., Brackett, J . K., Lynch, M . J., Hoh, J . T., and Matthews, K . S . (1991) J . Biol . Chem . 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Kramer, H., and Muller-Hill, B . (1991) New Biol . 3, 57-62) . To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed . The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor . The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography . Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins . Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion . Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH . Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer . "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed . The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored . This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure.

Biochim Biophys Acta, 1992 Jul 15, 1131(3), 307 - 10
A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells; Ke Y et al.; Human basic fibroblast growth factor has been expressed in Escherichia coli cells at a level of 2-3 mg/l culture, using a rapid procedure which requires only simple DNA manipulative work . The recombinant material has the same potency as natural basic fibroblast growth factor from bovine pituitary glands.

J Immunol, 1992 Jul 15, 149(2), 454 - 60
Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity; Moser M et al.; Aspergillus fumigatus secretes an 18-kDa nonglycosylated IgE-binding protein . This protein was previously shown to be a ribotoxin, like alpha-sarcin and mitogillin . A 686-bp long A . fumigatus cDNA encoding an 18-kDa ribotoxin was cloned and expressed in Escherichia coli as a fusion protein with six adjacent histidines (rAsp f I/a) . rAsp f I/a was purified to homogeneity by Ni(2+)-chelate affinity chromatography and refolded . The recombinant protein was enzymatically active resulting in the cleavage of 28S rRNA within a universally conserved region . rAsp f I/a was cytotoxic for EBV immortalized or PHA stimulated human PBMC . Furthermore, rAsp f I/a was recognized by murine mAb made against an 18-kDa ribotoxin . IgE of individuals allergic to A . fumigatus bound to rAsp f I/a as shown by ELISA, dot blots, and Western blots . rAsp f I/a elicited positive immediate type I skin reactions in individuals allergic to A . fumigatus but not in healthy control individuals . The results show that rAsp f I/a has similar functional characteristics when compared to the native 18-kDa ribotoxin . rAsp f I/a expressed in E . coli can therefore be used as a standardized Ag/allergen for serologic and clinical diagnosis of A . fumigatus-associated diseases.

Eur J Biochem, 1992 Jul 15, 207(2), 463 - 70
Cloning, sequencing and in vivo expression of genes encoding the F0 part of the sodium-ion-dependent ATP synthase of Propionigenium modestum in Escherichia coli; Kaim G et al.; A DNA fragment containing the genes encoding subunits of the F0 part of the sodium-translocating ATPase of Propionigenium modestum was cloned in Escherichia coli and sequenced . The predicted amino acid sequences of subunits a, b and c of the P . modestum ATPase were compared with those of the corresponding subunits of proton-translocating ATPases from other bacteria and chloroplasts . Deletion mutants of E . coli, lacking different genes for ATPase subunits, were transformed with a recombinant plasmid, containing the genes for the subunits a, c, b, delta and part of alpha of the ATPase of P . modestum . Functionally reconstituted ATPase activity could be demonstrated for the transformants . The identity of the vector containing P . modestum genes was verified by restriction analysis of plasmid DNA.

J Biol Chem, 1992 Jul 15, 267(20), 14175 - 82
Protein-protein interaction studied by site-directed mutagenesis . Characterization of the annexin II-binding site on p11, a member of the S100 protein family; Kube E et al.; p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II . The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K . (1988) EMBO J . 7, 2435-2442) . We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography . To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis . Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding . It is located in the C-terminal extension, following the second distorted EF-hand . Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II . Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect . Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation.

Biochem Biophys Res Commun, 1992 Jul 15, 186(1), 483 - 90
A gradient in expression of the Escherichia coli heat-stable enterotoxin receptor exists along the villus-to-crypt axis of rat small intestine; Cohen MB et al.; Binding of Escherichia coli heat-stable enterotoxin to its receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this receptor binds an endogenous ligand . In order to characterize the expression of the heat-stable enterotoxin receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum . Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity . Northern blots of total RNA identified a single heat stable enterotoxin receptor transcript (3.8 kb), predominantly in the villus cell fractions . In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis . Expression of this receptor was greatest after enterocytes leave the proliferative cycle and enter villi . This pattern of gene and protein expression may reflect a role of this receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis.

J Biol Chem, 1992 Jul 15, 267(20), 14429 - 35
Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli; Graves RJ et al.; Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups . In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites . Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases . The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination . The release of 5'-terminal dRp by crude extracts of E . coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction.

J Biol Chem, 1992 Jul 15, 267(20), 13998 - 4004
In vitro expression of thyroxine-binding globulin (TBG) variants . Impaired secretion of TBGPRO-227 but not TBGPRO-113; Janssen OE et al.; Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood . Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency . In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes . Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed . TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding . TBG-CD5 had altered electrophoretic mobility and did not bind T4 . TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly . Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M . The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.

J Biol Chem, 1992 Jul 15, 267(20), 13879 - 83
The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1; Loya S et al.; The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280 . In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis . Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified . In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme . The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested . The double cysteine mutant RT, on the other hand, exhibits several unique features . First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes . This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar . Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT . The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs . Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs . The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins.

J Biol Chem, 1992 Jul 15, 267(20), 13823 - 9
Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli; Hsu MY et al.; msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B . C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M . (1989) Science 243, 1033-1038) . The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT) . These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator . msDNA-Ec67 was produced only when cells were transformed with both plasmids . In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT . Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound . This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue . The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA . DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder . The DNA sequence determined from this ladder completely agreed with the msDNA sequence . The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase . This clearly demonstrates that RNA but not DNA is responsible for the msDNA production . A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model . In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT . Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R . A., and Yee, T . (1991) J . Biol . Chem . 266, 14497-14503).

Biochem J, 1992 Jul 15, 285 ( Pt 2), 503 - 6
Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids; Mizushima T et al.; The DNA relaxation activity of Escherichia coli DNA topoisomerase I in vitro was greatly inhibited by cardiolipin . Inhibition also occurred to some extent with phosphatidylglycerol from egg yolk . Analysis with synthetic phospholipid revealed that phosphatidylglycerol containing unsaturated fatty acids exhibited a strong inhibitory effect, whereas inhibition by phosphatidylglycerol containing saturated fatty acids was weak . Phosphatidylethanolamine showed no inhibitory effect . Chlorpromazine, which interacts with phospholipids, suppressed the inhibitory effect of cardiolipin . Cardiolipin and phosphatidylglycerol with unsaturated fatty acid precipitated topoisomerase I even at low concentrations, whereas phosphatidylglycerol from egg yolk and a synthetic phosphatidylglycerol containing saturated fatty acids precipitated this enzyme only at high concentrations . One-third of the total topoisomerase I in E . coli was found in the membrane fraction . Treatment of E . coli cells with chlorpromazine resulted in relaxation of plasmid DNA . This DNA relaxation was not observed in a topA mutant, suggesting that this relaxation by chlorpromazine in vivo is catalysed by topoisomerase I.

Eur J Biochem, 1992 Jul 15, 207(2), 479 - 85
Escherichia coli Rep protein and helicase IV . Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro; Yancey-Wrona JE et al.; Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichia coli and characterized . Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E . coli single-stranded DNA binding protein (SSB) . The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates . Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length . However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate . At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate . The addition of E . coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein . However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold . In addition, ionic strength conditions which stabilize duplex DNA (i.e . addition of MgCl2 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction . Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA . Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.

Eur J Biochem, 1992 Jul 15, 207(2), 435 - 39
Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen; Kumar S et al.; The thioredoxin system, comprising NADPH, thioredoxin reductase and thioredoxin reduces protein disulfides via redox-active dithiols . We have discovered that sodium selenite is a substrate for the thioredoxin system; 10 microM selenite plus 0.05 microM calf thymus thioredoxin reductase at pH 7.5 caused a non-stoichiometric oxidation of NADPH (100 microM after 30 min) . In contrast, thioredoxin reductase from Escherichia coli showed no direct reaction with selenite, but addition of 3 microM E . coli thioredoxin also resulted in non-stoichiometric oxidation of NADPH, consistent with oxidation of the two active-site thiol groups in thioredoxin to a disulfide . Kinetically, the reaction was complex with a lag phase at low selenite concentrations . Under anaerobic conditions the reaction stopped after 1 mol selenite had oxidized 3 mol NADPH; the admission of air then resulted in continued consumption of NADPH consistent with autooxidation of selenium intermediate(s) . Ferricytochrome c was effectively reduced by calf thymus thioredoxin reductase and selenite in the presence of oxygen . Selenite caused a strong dose-dependent inhibition of the formation of thiol groups from insulin disulfides with either the E . coli or calf-thymus thioredoxin system . Thus, under aerobic conditions selenite catalyzed, NADPH-dependent redox cycling with oxygen, a large oxygen-dependent consumption of NADPH and oxidation of reduced thioredoxin inhibiting its disulfide-reductase activity.

J Biol Chem, 1992 Jul 15, 267(20), 14470 - 6
Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor; Moss SJ et al.; Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system . These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins . Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology . Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases . We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) . The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC . PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide . Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases . The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA . The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide . Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC . The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit . The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides . Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit . No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC . These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1992 Jul 15, 267(20), 14418 - 23
Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K); Pickart CM et al.; Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation . Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next . During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination) . Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59 . The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin . The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59 . Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons . Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase . These findings should assist in the design of future studies concerning E2(25K) structure and function.

J Biol Chem, 1992 Jul 15, 267(20), 14366 - 72
Isolation and characterization of a gene encoding rat nucleoside diphosphate kinase; Ishikawa N et al.; Two overlapping genomic clones for a rat nucleoside diphosphate kinase (NDP kinase) have been isolated and characterized . Complete sequencing of the genomic segment including the whole coding region for the enzyme revealed that the gene consists of four exons spanning 5.5 kilobase pairs . Primer extension analyses and ribonuclease protection assays indicated that the transcription may start from multiple sites with the major initiation site at 3 base pairs upstream from the translation initiation site, Met-1 . Neither CAAT-box nor TATA-box could be assigned for each transcription initiation site, whereas five putative Sp1-binding sites (GC-boxes) were present in the 5'-flanking region . These features of the NDP kinase gene represent those of housekeeping genes . In genomic Southern blotting using a full-length rat NDP kinase cDNA as a probe, many positively hybridized fragments were detected . In support of this, five possible processed pseudogenes were identified in different DNA segments although many other NDP kinase-related genomic fragments remained to be characterized . These results demonstrate that the NDP kinase gene may consist of a multiple gene family.

J Biol Chem, 1992 Jul 15, 267(20), 14151 - 6
Structural requirements for the growth factor activity of the amino-terminal domain of urokinase; Rabbani SA et al.; High molecular weight urokinase-type plasminogen activator (uPA) in which proteolytic activity was inactivated (diisopropyl fluorophosphate (DFP)-uPA), its amino-terminal fragment (ATF, amino acids (aa) 1-143), and fucosylated and defucosylated growth factor domains (GFD, aa 4-43) were tested for growth-promoting effects and binding in human SaOS-2 osteosarcoma cells and U-937 lymphoma cells . DFP-uPA, ATF, and both the fucosylated and defucosylated GFD were capable of competing with 125I-ATF for binding to both SaOS-2 and U-937 cells . DFP-uPA, ATF, and fucosylated GFD were also mitogenic in SaOS-2 cells and increased cell numbers . However, defucosylated GFD was nonmitogenic in SaOS-2 cells and did not stimulate cell proliferation, even though it bound to these cells in a manner equivalent to the fucosylated GFD . A nonglycosylated high molecular weight uPA expressed and purified from Escherichia coli inhibited 125I-ATF binding to SaOS-2 cells but was also nonmitogenic . No mitogenic activity was observed in U-937 cells treated with the uPA forms capable of eliciting a mitogenic response in SaOS-2 cells . Proteolytically prepared kringle domain (aa 47-135) and low molecular weight uPA (aa 144-411) did not compete for 125I-ATF binding and did not elicit any mitogenic response in either of the cell lines tested . In addition, tissue plasminogen activator (tPA), which has been shown to be homologous to uPA in its growth factor domain and is also fucosylated, did not inhibit 125I-ATF binding nor elicit any mitogenic response . These results demonstrate that the GFD, implicated in binding to the uPA receptor, is also responsible for growth factor like activity in SaOS-2 cells and that the fucosylation at Thr18 within this domain may serve as a molecular trigger in eliciting this response.

J Biol Chem, 1992 Jul 15, 267(20), 13811 - 4
Insulin receptor protein-tyrosine phosphatases . Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain; Hashimoto N et al.; A number of protein-tyrosine phosphatase(s) (PTPases) have been shown to dephosphorylate the insulin receptor in vitro; however, it is not known whether any individual PTPase has specificity for certain phosphotyrosine residues of the receptor that regulate its intrinsic tyrosine kinase activity . We evaluated the deactivation of the insulin receptor kinase by three candidate enzymes that are expressed in insulin-sensitive rat tissues, including the receptor-like PTPases LAR and LRP, and the intracellular enzyme, PTPase1B . Purified insulin receptors were activated by insulin and receptor dephosphorylation, and kinase activity was quantitated after incubation with recombinant PTPases from an Escherichia coli expression system . When related to the level of overall receptor dephosphorylation, LAR deactivated the receptor kinase 3.1 and 2.1 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.03) . To assess whether these effects were associated with preferential dephosphorylation of the regulatory (Tyr-1150) domain of the receptor beta-subunit, we performed tryptic mapping of the insulin receptor beta-subunit after dephosphorylation by PTPases . Relative to the rate of initial loss of 32P from receptor C-terminal sites, LAR dephosphorylated the Tris-phosphorylated Tyr-1150 domain 3.5 and 3.7 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.01) . The accelerated deactivation of the insulin receptor kinase by LAR and its relative preference for regulatory phosphotyrosine residues further support a potential role for this transmembrane PTPase in the physiological regulation of insulin receptors in intact cells.

Biochemistry, 1992 Jul 14, 31(27), 6272 - 8
Co2+ as a shift reagent for 35Cl NMR of chloride with vesicles and cells; Shachar-Hill Y et al.; Applications of high-resolution 35Cl NMR to the study of chloride in vivo and in vesicles have hitherto been limited by problems of NMR detectability and of resolving internal from external signals . We have characterized the effects of Co2+ on the 35Cl resonance of Cl- in solution and have shown that when added to suspensions of lipid vesicles, Co2+ shifts the 35Cl signal of the extravesicular Cl-, allowing clear resolution and quantitation of two peaks . We have assigned these signals to chloride inside and outside the vesicles . The spectra do not change over a 90-min period, demonstrating the stability of the vesicles in the presence of Co2+ . This technique is shown to be applicable to red blood cell ghosts, where intravesicular and extravesicular chloride signals were separated and measured and chloride/sulfate exchange through the band 3 anion transport protein A was followed . In two plant species (an alga and a higher plant), an intracellular Cl- signal can be observed and resolved from the extracellular signal . The intracellular transportable chloride was found to be fully NMR-visible (+/- 5%) in the algal cells . The high steady-state levels of Cl- seen in the alga were consistent with previous work using 36Cl- labeling on a related species {Doblinger, R., & Tromballa, H.W . (1982) Planta 156, 10-15} . Successive spectra acquired after adding Co2+ to Chlorella cells under deenergizing conditions allow us to follow the time course of movement of Cl- out of the cells.

Biochemistry, 1992 Jul 14, 31(27), 6219 - 23
Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase; Haselbeck RJ et al.; The cDNA for porcine mitochondrial NADP-specific isocitrate dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction . Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide . Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence . The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific isocitrate dehydrogenase and to be immunoreactive with an antiserum against the yeast protein . Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme . The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent isocitrate dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-isocitrate site of the procaryotic enzyme.

Biochemistry, 1992 Jul 14, 31(27), 6285 - 90
Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure; Norwood TJ et al.; Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented . A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol . Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved . Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure . This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.

Biochemistry, 1992 Jul 14, 31(27), 6279 - 84
Potential of 13C and 15N labeling for studying protein-protein interactions using Fourier transform infrared spectroscopy; Haris PI et al.; In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy . In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance . Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band . However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band . This opens up windows in different regions of the infrared spectrum . Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl . This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy . With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible . These could include, among others, enzyme-substrate and protein-ligand interactions.

Biochim Biophys Acta, 1992 Jul 13, 1122(1), 77 - 84
Overproduction, purification and characterization of SecD and SecF, integral membrane components of the protein translocation machinery of Escherichia coli; Matsuyama S et al.; SecD and SecF proteins were overproduced by means of recombinant DNA technology . Immunoblot and amino-acid sequencing analysis revealed that the overproduced proteins are SecD and SecF . The SecD- or SecF-overproduced membrane fraction was subjected to differential solubilization . The SecD protein was then purified through ion-exchange and size-exclusion chromatographies . The SecF protein was purified through size exclusion chromatography . Proteoliposomes reconstituted from the purified SecD and SecF together with SecE and SecY were used to analyze the translocation activity . SecD and SecF did not exhibit significant effects on the translocation activity of proteoliposomes . The amounts of SecD and SecF in overproducers were determined densitometrically on a stained SDS gel and their overproduction (fold) was determined by means of immunoblot analysis . Then the number of these molecules in one normal cell were estimated . From these numbers, together with those of other Sec proteins, the number of the translocation machinery existing in one Escherichia coli cell was inferred to be around 500.

J Clin Endocrinol Metab, 1992 Jul, 75(1), 121 - 6
Identification of localized autoantibody epitopes in thyroid peroxidase; Maastricht J et al.; Recent reports have disagreed on the nature of the autoantibody epitopes in thyroid peroxidase (TPO) . We used immunoprecipitation of recombinant human TPO constructs to determine if localized autoantibody binding sites exist in this autoantigen . In vitro transcription and translation of TPO cDNA fragments yielded 35S-labeled products consisting of either full-length protein (933 amino acids) or N-terminal peptides of 631, 455, and 120 amino acids . Immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the Hashimoto's sera consistently precipitated the full-length and the 631 amino acid products, but not the shorter N-terminal peptides . An additional construct resulting in a full-length TPO peptide with an internal deletion of amino acids 4-455 was also made, and this product was also precipitated by the Hashimoto's sera . A fusion protein consisting of maltose binding protein followed by amino acids 456-933 of human TPO was produced in Escherichia coli and subjected to Western blot analysis using the Hashimoto's sera . The Hashimoto's sera reacted with the MalTose binding protein TPO (MBP/TPO) fusion protein, but not a control fusion protein (MBP/LacZ alpha) . Together, these results indicate the presence of localized autoantibody epitopes in the portion of the human TPO molecule from amino acids 456 to 933, with at least one binding site located between amino acids 456 and 631.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3471 - 7
Escherichia coli promoters: neural networks develop distinct descriptions in learning to search for promoters of different spacing classes; O'Neill MC; Back-propagation neural networks were trained to recognize promoter sequences of each of the three major spacing classes found in E . coli . These networks were trained with the object of maximizing their ability to generalize while maintaining the level of false positive identifications at a fraction of 1 percent . These objectives were generally met . Networks for the 16 base spacing class captured between 78 and 100% of previously unseen promoters in different tests; networks for the 17 base class identified 97% of the test promoters; networks for the 18 base class identified 79% of the test promoters . A tandem poll of networks for all three spacing classes produced a cumulative false positive level of less than 0.5% . In each case, the weight matrices used by the networks in their classification were analyzed to determine the relative weight assigned to the occurrence of a given base at a given position within the promoter . In this fashion, an approximate description of the network's definition of the promoter can be obtained.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3391 - 6
A comparison of the DNA bending activities of the DNA binding proteins CRP and TFIID; Gaston K et al.; Protein-induced DNA bending is of importance in the formation of complex nucleoprotein assemblies such as those involved in the initiation of DNA replication or transcription initiation . We have compared the DNA bending characteristics of the Escherichia coli cyclic AMP receptor protein (CRP or CAP), an archetypal DNA bending protein, to those of TFIID, the eukaryotic TATA-element binding transcription factor . By altering the helical phasing between a CRP binding site and the E . coli melR promoter we have mapped a DNA sequence-directed bend in the downstream region of the promoter . This intrinsic DNA bend may be important in the regulation of the melR promoter by CRP in vivo . Gel retardation assays and DNAse I footprinting show that human TFIID binds to the melR promoter - 10 region . Taking advantage of this fact, and using the CRP-induced DNA bend as a standard, we have employed phase sensitive detection to show that the DNA bend angle induced by TFIID is far less than that induced by CRP . Further evidence to support this conclusion comes from a comparison of the relative mobilities of CRP-DNA and TFIID-DNA complexes . These results place limits on the role of any DNA bending induced by TFIID alone in the initiation of transcription.

Biochem Pharmacol, 1992 Jul 7, 44(1), 137 - 48
Effects of interleukin-6 on cytochrome P450-dependent mixed-function oxidases in the rat; Chen YL et al.; Intravenous treatment of male rats with recombinant human interleukin-6 (rhIL6) at 50, 100 and 200 micrograms/kg (corresponding to 4, 8 and 16 x 10(4) U/animal, respectively) reduced the activities of hepatic microsomal cytochrome P450-dependent monoxygenases to varying degrees . Ethylmorphine-N-demethylase activity fell to 53% of control values, an effect similar to that induced by 2.5 mg/kg Escherichia coli lipopolysaccharide (LPS) . Ethoxycoumarin-O-deethylase activity was also sensitive to inhibition, whereas IL6 had little effect on the activities of other P450-dependent enzymes, including ethoxyresorufin-O-deethylase . Pentoxyresorufin dealkylase activity, which is representative of the cytochrome P450 IIB 1/2 subfamily, was unaffected by IL6 whereas LPS reduced it to 33.7% of control values . Another hepatocyte-related parameter, serum concentration of alpha 1-acid glycoprotein (AGP), was increased by up to 3.5-fold over baseline by IL6 and 10-fold by LPS . Recombinant human interleukin-1 beta (rhIL1 beta) (10 micrograms/kg, corresponding to 5 x 10(4) U/rat) and recombinant human tumor necrosis factor alpha (rhTNF) (150 micrograms/kg corresponding to 24 x 10(4) U/rat) were both as potent as LPS (2.5 mg/kg) in increasing serum AGP levels and reducing hepatic microsomal monoxygenase activities . IL6 did not potentiate the effects of rhIL1 beta . Hepatic microsomal glucuronyltransferase activities were little affected by LPS and unaffected by rhIL6 . Finally, rhIL6 was more potent after i.p . injection than after i.v . or s.c . injection . These results suggest that the effects of LPS, TNF and IL1 on the mixed-function oxidase system in vivo may be due partly to an induction of IL6 in vivo . The different sensitivities of the enzymes to IL6 but not to IL1 or TNF may be due to the involvement of two distinct mechanisms.

Biochemistry, 1992 Jul 7, 31(26), 6166 - 74
Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions; Wei TF et al.; The Escherichia coli single-strand binding (SSB) protein is an essential protein involved in DNA replication, recombination, and repair processes . The tetrameric protein binds to ss nucleic acids in a number of different binding modes in vitro . These modes differ in the number of nucleotides occluded per SSB tetramer and in the type and degree of cooperative complexes that are formed with ss DNA . Although it is not yet known whether only one or all of these modes function in vivo, based on the dramatically different properties of the SSB tetramer in these different ss DNA binding modes, it has been suggested that the different modes may function selectively in replication, recombination, and/or repair . The transitions between these different modes are very sensitive to solution conditions, including salt (concentration, as well as cation and anion type), pH, and temperature . We have examined the effects of multivalent cations, principally the polyamine spermine, on the SSB-ss poly(dT) binding mode transitions and find that the transition from the (SSB)35 to the (SSB)56 binding mode can be induced by micromolar concentrations of polyamines as well as the inorganic cation Co(NH3)6(3+) . Furthermore, these multivalent cations, as well as Mg2+, induce the binding mode transition by binding cooperatively to the SSB-poly(dT) complexes . These observations are interesting in light of the fact that polyamines, such as spermidine, are part of the ionic environment in E . coli and hence these cations are likely to affect the distribution of SSB-ss DNA binding modes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jul 7, 31(26), 6089 - 97
EcoRV restriction endonuclease: communication between DNA recognition and catalysis; Vermote CL et al.; A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins . The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188 . In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence . But neither mutation affected the ability of the protein to bind to DNA . In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme . In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type . In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type . When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme . This was the reason for their low activities with Mg2+ as the cofactor . The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.

Biochem Pharmacol, 1992 Jul 7, 44(1), 17 - 24
The metabolism of glyceryl trinitrate to nitric oxide in the macrophage cell line J774 and its induction by Escherichia coli lipopolysaccharide; Salvemini D et al.; The metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) was studied in the mouse macrophage cell line J774 and in the human monocytic cell line U937 in the absence or presence of Escherichia coli lipopolysaccharide (LPS) . Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in J774 cells . In addition, NO produced from GTN by cells or by cellular fractions was measured as nitrite (NO2-) one of its breakdown products . J774 cells (1.25 x 10(5) cells) treated with indomethacin (10 microM) enhanced the platelet inhibitory activity of GTN (22-352 microM) but not that of sodium nitroprusside (4 microM) . This effect was abrogated by co-incubation with oxyhaemoglobin (oxyHb, 10 microM) indicating release of NO from GTN . U937 cells (up to 60 x 10(5)) did not metabolize GTN to NO . LPS (0.5 micrograms/mL for 18 hr) enhanced at least 2-fold the capacity of J774 cells but not that of U937 cells to form NO from GTN and this enhancement was attenuated when cycloheximide (10 micrograms/mL) was incubated together with LPS . In the absence of LPS stimulation, cycloheximide had no effect . Furthermore, when incubated with GTN (200 microM), J774 cells treated with LPS released more NO from GTN as indicated by a 3-fold greater increase in their level of cGMP which was prevented by oxyHb (10 microM) . Incubation of J774 cells with GTN (75-600 microM) for 30 min led to a concentration-dependent increase in NO2- which was substantially reduced when the cells were boiled . The microsomal fraction was more potent than the cytosol in producing NO2- from GTN (1.2-2.4 mM) . Release of NO2- from GTN by J774 cells was not affected by treating the cells with the NO synthase inhibitor, NG-monomethyl-L-arginine (MeArg, 300 microM) . In J774 cells made tolerant to GTN, potentiation of the anti-platelet effects of GTN (11-352 microM) and release of NO2- from GTN was reduced . Thus, J774 cells but not U937 cells convert GTN to NO . This enzymic pathway (present mainly in the microsomal fraction of the J774 cells) is induced by LPS and is not regulated by endogenous NO released from L-Arg by the enzyme NO synthase . Furthermore, when compared to normal cells, tolerant J774 cells metabolize GTN to NO less effectively as assessed by a reduced capacity to potentiate the anti-platelet effect of GTN and to release NO2-.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1992 Jul 7, 31(26), 6103 - 10
Functional analysis of chimeric proteins constructed by exchanging homologous domains of two P-glycoproteins conferring distinct drug resistance profiles; Dhir R et al.; P-Glycoproteins (P-gps) encoded by the mouse mdr1 and mdr3 (Phe939, mdr3F) genes confer distinct drug resistance profiles . While the mdr1 and mdr3F clones confer comparable levels of vinblastine (VBL) resistance, mdr3F confers actinomycin D (ACT) resistance levels 2-fold greater than mdr1, while mdr1 confers resistance to colchicine at levels 7-fold greater than mdr3F . We wished to identify in chimeric proteins discrete protein domains responsible for the distinct drug resistance profiles of mdr1 and mdr3F . Homologous protein domains were exchanged in hybrid cDNA clones, and the specific drug resistance profiles conferred by chimeric proteins were determined in stably transfected cell clones expressing comparable amounts of wild-type or chimeric P-gps . Immunoblotting experiments showed that all chimeras were found expressed in membrane-enriched fractions of transfected cell clones and all conveyed cellular drug resistance at levels above the background of nontransfected drug-sensitive LR73 cells . For VBL, all chimeric constructs were found to convey similar levels of resistance . For COL and ACT, the levels of resistance conferred by the various chimeras were heterogeneous, being similar to either the parental mdr1 or the parental mdr3F clones, or in many cases being intermediate between the two . The preferential COL and ACT resistance phenotypes of mdr1 and mdr3F, respectively, did not segregate in chimeric proteins with any specific protein segment . Taken together, our results suggest that the preferential drug resistance phenotypes encoded by the mdr1 and mdr3F clones implicate complex interactions between the two homologous halves of the respective P-gp.

Biochemistry, 1992 Jul 7, 31(26), 6045 - 56
Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem; Gonzalez JC et al.; In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases . Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem . We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins . We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase . Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution . We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies . In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide . We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity . While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.

Biochemistry, 1992 Jul 7, 31(26), 6111 - 8
Vesicle fusion in protein transport through the Golgi in vitro does not involve long-lived prefusion intermediates . A reassessment of the kinetics of transport as measured by glycosylation; Hiebsch RR et al.; The well-characterized cell-free assay measuring protein transport between compartments of the Golgi {Balch, W . E., Dunphy, W . G., Braell, W . A., & Rothman, J . E . (1984) Cell 39, 405-416} utilizes glycosylation of a glycoprotein to mark movement of that protein from one Golgi compartment to the next . Glycosylation had been thought to occur immediately after vesicles carrying the glycoprotein fuse with their transport target . Therefore, the kinetics of glycosylation were taken to reflect the kinetics of vesicle fusion . We previously isolated and raised monoclonal antibodies against a protein (the prefusion operating protein, POP) which is required in this assay at a step after vesicles have apparently been formed and interacted with the target membranes, but long before glycosylation takes place . This was therefore presumed to be a reaction involving targeted but unfused vesicles . Here we report that POP is identical to uridine monophosphokinase, as revealed by molecular cloning . We show that POP is not active in transport per se but instead enhances the glycosylation used to mark transport . This indicated that, contrary to previous assumptions, glycosylation might lag significantly behind vesicle fusion . We directly show this to be true . This alters the interpretation of several earlier studies . In particular, the previously reported existence of a late, prefusion intermediate, the "NEM-resistant intermediate", can be seen to be due to effects on glycosylation and not indicative of true fusion events.

Mol Cell Biochem, 1992 Jul 6, 113(1), 55 - 61
High affinity DNA-microtubule associated protein interaction; Marx KA; We have isolated the MAP/tau proteins from twice-cycled chick brain microtubule preparations and demonstrated that they are responsible for the nitrocellulose DNA binding activity we and others have measured . Using the isolated MAP/tau proteins we then measured the apparent affinity constant K(app) for the homologous chick DNA interaction and found evidence for two equilibrium affinity classes-a K(app) = 6 x 10(7) M-1, responsible for the bulk of the DNA binding activity and a small (less than 10%) higher affinity K(app) = 10(8) - 10(9) M-1, likely due to sequence specific binding protein species . Using the same chick brain MAP-tau protein, a heterologous interaction with D . melanogaster DNA, was found to possess just the lower affinity class-K(app) = 2 x 10(7) M-1 . Under stringent binding conditions we carried out equilibrium nitrocellulose filter binding experiments in a ternary reaction mixture at constant MAP/tau protein and 35S radiolabelled chick DNA concentration using increasing and excess concentrations of competitor DNAs of different sources . The order of competitor strengths found was-chick DNA greater than mouse DNA greater than D . melanogaster = E . coli . DNA . These data and specifically the homologous DNA: protein case being the strongest competitor corroborate our previous studies using total microtubule protein and provide new evidence for a conserved interaction of a small DNA sequence class with MAP/tau protein species . Moreover, these data allow us to conclude that the conserved DNA sequence: MAP/tau protein interactions do not critically depend upon any energetic feature co-involving tubulin for their properties since tubulin is absent from these preparations.

J Immunol Methods, 1992 Jul 6, 151(1-2), 299 - 306
A method for the detection of serologically crossreacting antigens both within and between protein mixtures: the western cross blot; Hammerl P et al.; The method described in the present paper permits the detection of antigenically related proteins within or between different antigen mixtures . For this purpose two separate SDS-polyacrylamide gels are run (the antigen mixture is applied to the entire width of the gel) and each gel is blotted on to a separate nitrocellulose paper . One sheet ('donor sheet') is incubated with antiserum and placed on to the other blot ('receptor sheet') so that the antigen bands are perpendicular to each other . Subsequently the antibodies from the donor sheet are blotted on to the receptor sheet in alkaline borate buffer containing 1 M NaSCN, which dissociates antigen-antibody complexes . After the removal of the donor sheet the receptor sheet containing the transblotted antibodies is equilibrated in phosphate-buffered saline in order to reconstitute the binding conditions . Antibodies which have bound to receptor antigens during this equilibration period are detected by the use of a peroxidase labelled 2nd antibody . Because each band on the donor sheet crosses each band on the receptor sheet during the transfer, the antibodies from each band of the donor sheet are able to react with each antigen band of the receptor sheet . The validity of the method was established by demonstrating the crossreactivity of a polyclonal antiserum against the intact bovine gamma globulin molecule with the heavy and light chain subunits (and partial reduction products of the molecule) and the absence of crossreactivity between heavy and light chains . In a second experiment crossreacting antigens of different molecular weight were detected within several strains of Escherichia coli . In addition, comparisons of different strains of Escherichia coli revealed crossreacting antigens of identical as well as of different molecular weight.

J Immunol Methods, 1992 Jul 6, 151(1-2), 177 - 89
The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins; Veldhoven CH et al.; A characteristic of patients with autoimmune diseases such as Sjogren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation . In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed . Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E . coli as non-fusion proteins . These were purified to homogeneity using two different purification protocols . With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed . 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA . Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA . The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B) . Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test . From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins . For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.

FEBS Lett, 1992 Jul 6, 305(3), 257 - 9
Is thioredoxin the physiological vitamin K epoxide reducing agent?
Preusch PC.
E . coli thioredoxin plus thioredoxin reductase have previously been shown to replace dithiothreitol as the electron donor for mammalian liver microsomal vitamin K epoxide reduction in vitro . Such activity is dependent on detergent disruption of the microsomal membrane integrity . A previously characterized salicylate-inhibitable pathway for electron transfer from endogenous cytosolic reducing agents to the microsomal epoxide reducing warfarin-inhibitable enzyme is not inhibited by known alternate substrates and inhibitors of the thioredoxin system nor by antibodies against thioredoxin.

FEBS Lett, 1992 Jul 6, 305(3), 177 - 80
A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding; Puri NK et al.; Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urea or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC) . Circular dichroism (CD) analysis of the secondary (2 degrees) structure of the urea- and GnHCl-solubilised rPGH showed the absence of alpha-helical content with the majority of the molecule existing in a 'random coil' structure . In contrast, the CTAC-solubilised rPGH displayed significant starting 2 degrees structure (10-15% alpha helix; 30-40% beta structure) . The three rPGH preparations were refolded in vitro against weak urea . GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's . We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2 degrees structure of rPGH, particularly alpha-helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.

FEBS Lett, 1992 Jul 6, 305(3), 167 - 70
Characterisation of a near infra-red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high-spin ferrous haem of the binuclear site; Ingledew WJ et al.; The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA . In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem . The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm . This region in cytochrome aa3 is dominated by the CuA absorption . The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme . The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.

J Biol Chem, 1992 Jun 25, 267(18), 12761 - 6
Purification and properties of the F sex factor TraD protein, an inner membrane conjugal transfer protein; Panicker MM et al.; Using a traD overexpression plasmid, we purified the F sex factor TraD protein in milligram quantities . The purified protein has an apparent molecular weight of 82,000 and an amino acid composition rich in acidic residues . Using specific antibodies, TraD was localized to the inner membrane of F+ cells under conditions where it is produced in physiologically normal amounts . Furthermore, the protein was soluble only in the presence of detergents, but there is evidence that the carboxyl terminus is water-soluble . The purified protein shows pH-sensitive binding to DNA cellulose columns.

J Mol Biol, 1992 Jul 5, 226(1), 7 - 13
Molecular interactions in myosin assembly . Role of the 28-residue charge repeat in the rod; Atkinson SJ et al.; We have used internal deletions of multiples of seven residues to change the phase of the 28-residue charge repeat in a light meromyosin cDNA construct expressed in Escherichia coli . The solubility behaviour of these mutants was similar to that of the wild-type material, but the molecular packing in the aggregates formed at low ionic strength was different . Whereas wild-type material formed paracrystals in which molecules were in close contact over most of their length, molecules in the paracrystals formed by the mutants were in close contact for only a short distance, which was just short enough to exclude the deletion from the overlap . These data indicate that, although the 28-residue charge periodicity is important in myosin molecular interactions, it is probably not the major driving force for myosin assembly and instead influences the detailed axial stagger of the interacting molecules.

J Mol Biol, 1992 Jul 5, 226(1), 69 - 83
Isorepressor of the gal regulon in Escherichia coli; Weickert MJ et al.; Inducible overexpression of the Escherichia coli gal operon in the absence of the Gal repressor is known as ultrainduction . The requirement of induction can be eliminated by mutation of a new locus, galS, resulting in constitutive and ultrainduced levels of gal expression . Characterization of the galS gene and its product has revealed an isorepressor of the gal regulon . The Gal isorepressor is a protein of 346 amino acid residues whose amino acid sequence and cellular function, as described here, are very similar to that of Gal repressor, encoded by the galR gene . Transcription from different promoters of the gal regulon, galP1, galP2 and mglP, was examined by primer extension and reverse transcription of mRNA isolated from strains containing mutations in galR and/or galS . In strains containing a galS mutation, overexpression of gal message occurred only in the presence of inducer, while mgl message was constitutively derepressed . The galS mutation also constitutively derepressed an mglA::lacZ fusion, demonstrating that GalS is the mgl repressor . A potential operator site in the mgl promoter was identified at a position analogous to OE in gal . Thus, the gal and mgl operons constitute a regulon . Crosstalk, temporal action, induction spectrum or heteromer formation between repressor and isorepressor may help co-ordinate high affinity galactose transport and galactose utilization.

J Mol Biol, 1992 Jul 5, 226(1), 15 - 22
Atomic interactions in protein-carbohydrate complexes . Tryptophan residues in the periplasmic maltodextrin receptor for active transport and chemotaxis; Spurlino JC et al.; We have refined the 1.9 A resolution crystal structures of two maltodextrin receptor mutants in which tryptophan residues 230 and 232 have been changed to alanine and compared these structures with the refined 1.7 A structure of the wild-type protein . In the wild-type structure, Trp230, which is located in the maltodextrin-binding groove, stacks against the B-face of the reducing sugar of the bound maltose . Trp232, which is located near the protein surface, does not participate directly in sugar binding . Relative to the wild-type structure, neither mutation caused a significant rearrangement in the overall protein structure or in the mode of binding maltose . Although the position once occupied by Trp230 remains empty, a new water molecule has moved near the void . In contrast, a new water molecule has entered into the space once occupied by Trp232 . Whereas one hydrogen bond is formed with the water molecule near the Trp230 void, no hydrogen bond is associated with the water molecule occupying the space vacated by Trp232 . The three van der Waals' contacts between Trp230 and maltose in the wild-type structure that are lost in the W230A mutation could contribute to the 12-fold decrease in ligand-binding activity of the mutant protein . The W232A mutation causes little change in binding activity . The structures of these mutant proteins also provide some insight into the complicated tryptophan fluorescence spectra of the maltodextrin binding-protein . The change in fluorescence due to the deletion of Trp230 can readily be explained as resulting directly from loss of Trp230 in the sugar-binding site . The change in fluorescence due to deletion of Trp232, however, is ascribed to the modification of local interactions mediated by the binding of maltodextrin since the tryptophan is not directly involved in any sugar-binding interaction.

J Mol Biol, 1992 Jul 5, 226(1), 127 - 39
Unwinding of heterologous DNA by RecA protein during the search for homologous sequences; Rould E et al.; The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates . The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA . To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa . In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA . This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes . Presynaptic filaments that were formed in the presence of ATP gamma S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase . The inhibition of heterologous contacts by 60 mM-NaCl or 5 mM-ADP resulted in a corresponding decrease in heterologous unwinding . In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA . These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.

J Biol Chem, 1992 Jul 5, 267(19), 13598 - 602
Bordetella pertussis adenylate cyclase toxin . Structural and functional independence of the catalytic and hemolytic activities; Sakamoto H et al.; The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity . We have constructed B . pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity . Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity . Furthermore, B . pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed . The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity . Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.

J Biol Chem, 1992 Jul 5, 267(19), 13564 - 72
Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits; Masterson C et al.; The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains . The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly . The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme . At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I . D., Robson, C . N., Atkinson, K . E., Hutton, L., and Emmerson, P . T . (1985) J . Biol . Chem . 260, 1224-1229) . At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.

J Biol Chem, 1992 Jul 5, 267(19), 13540 - 6
Characteristics of the F52 protein, a MARCKS homologue; Blackshear PJ et al.; A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC) . Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain . The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells . The F52 protein also was myristoylated in E . coli by co-expression with N-myristoyltransferase . A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4) . The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min . The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.

J Biol Chem, 1992 Jul 5, 267(19), 13440 - 5
Structural requirements for tRNA methylation . Action of Escherichia coli tRNA(guanosine-1)methyltransferase on tRNA(1Leu) structural variants; Holmes WM et al.; The Escherichia coli enzyme tRNA(m1G)methyltransferase, one of a group of post-transcription tRNA-modifying enzymes, shows remarkable specificity in selecting the tRNA species and the specific guanosine base to be methylated . To examine the structural basis of this specificity, we synthesized a total of 15 modifications of tRNA(1Leu) and measured their methylation reaction kinetics in vitro . Elimination of any one of the three tRNA side loops, the V loop, the T loop, or the D loop, reduced the Vmax for methylation by about 1 order of magnitude . Elimination of all three side loops reduced Vmax by about 2 orders of magnitude . Clearly, gross tRNA structure is important for full enzyme activity . At the bottom of the stem proximal to the anticodon loop, in the pair at positions 31-39, substitution of a G-C for a C-G, a change that should not weaken the helical structure, had little effect on Vmax or Km . However, substitution of a G for a C increased Vmax and Km, whereas substitution of a C for G sharply reduced Vmax and, to a lesser extent, Km . These results appear to be a consequence of the principle that purines are better than pyrimidines in the stacking of adjacent bases for stability . Stacking in the stem structure appears to be important for methylation enzyme activity . In the anticodon loop itself, changing a U to a C had little effect, but changing the G of the anticodon to a C reduced Vmax over 20-fold, demonstrating the importance of the presence of the anticodon G adjacent to the G being methylated for enzyme recognition.

J Biol Chem, 1992 Jul 5, 267(19), 13180 - 4
Escherichia coli dnaJ deletion mutation results in loss of stability of a positive regulator, CRP; Ohki R et al.; The dnaJ deletion mutant K7052(lambda dnaK) has a temperature-sensitive defect in the synthesis of beta-galactosidase . We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP . In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature . Introduction of a CRP over-producing plasmid into the dnaJ deletion mutant suppressed the defect of beta-galactosidase synthesis . The lower content of CRP in the mutant was found to result from extreme instability of the protein . These results strongly suggested that the heat shock protein dnaJ is involved in the stabilization (or degradation) of CRP.

J Mol Biol, 1992 Jul 5, 226(1), 59 - 68
How Lac repressor finds lac operator in vitro; Fickert R et al.; Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator . A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted . It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters . This assumption is wrong . Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions . Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator . A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor . It is presumably due to intersegment transfer involving lac operator-like sequences.

J Biol Chem, 1992 Jul 5, 267(19), 13127 - 30
Branched-chain alpha-ketoacid dehydrogenase kinase . Molecular cloning, expression, and sequence similarity with histidine protein kinases; Popov KM et al.; A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library . The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280 . The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E . coli expressing the protein . Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues . The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases . This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.

J Mol Biol, 1992 Jul 5, 226(1), 101 - 15
delta-Aminolevulinate couples cycA transcription to changes in heme availability in Rhodobacter sphaeroides; Schilke BA et al.; In this paper, the response of the transcriptional control region of the Rhodobacter sphaeroides cytochrome c2 gene, cycA, to intermediates in heme biosynthesis was studied . To determine if cycA transcription was regulated by heme availability, several precursors or analogs of tetrapyrroles were tested . Addition of delta-aminolevulinate (ALA), the first committed intermediate in heme biosynthesis, was shown to inhibit cycA transcription initiation at both the upstream and downstream promoter regions . In addition, an ALA auxotroph, which can grow in the presence of high levels of ALA, showed a 5 to 7-fold reduction in steady-state transcription from cycA::lacZYA operon fusions . To identify genetic elements responsible for negative regulation by ALA, trans-acting mutants with increased expression of cycA were isolated that were resistant to growth inhibition by the heme analog cohemin . These cohemin-resistant mutants (Chr) have elevated levels of several cycA transcripts and they contain cycA transcripts that had not previously been detected in wild-type cells . In addition, cycA transcription in the Chr mutants continues after the addition of ALA . Finally, we found that Chr mutants have increased ALA synthase activity, suggesting that synthesis of cytochrome c2 and ALA synthase are controlled by a common gene product whose activity has been modified in these mutants . A model is presented to explain how changes in tetrapyrrole intermediates could provide an effective signal to control both cycA transcription and ALA synthase synthesis in R . sphaeroides.

J Biol Chem, 1992 Jul 5, 267(19), 13629 - 35
A novel DNA helicase from calf thymus; Siegal G et al.; We report the purification and characterization of a novel DNA helicase from calf thymus tissue . This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it . The enzyme contains an intrinsic DNA-dependent ATPase activity . It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction . Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound . Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement . Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A . The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation . Direct {alpha-32P}ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site . In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.

Presse Med, 1992 Jul 4-11, 21(25), 1157 - 9
{Isolated dissecting aneurysm of the superior mesenteric artery . Dysplasia, a rare cause of mesenteric ischemia . Report of a case}; Marzelle J et al.; We report a case of successful surgical revascularization of the superior mesenteric artery in a patient presenting with intestinal ischaemia due to fibrodysplasia . This is a rare pathology, with 5 cases operated in our institution and only 16 cases reported in the literature . Angiography may show other sites of arterial dysplasia . In such cases, success depends on early surgical revascularization.

Science, 1992 Jul 3, 257(5066), 39 - 49
Chance and statistical significance in protein and DNA sequence analysis; Karlin S et al.; Statistical approaches help in the determination of significant configurations in protein and nucleic acid sequence data . Three recent statistical methods are discussed: (i) score-based sequence analysis that provides a means for characterizing anomalies in local sequence text and for evaluating sequence comparisons; (ii) quantile distributions of amino acid usage that reveal general compositional biases in proteins and evolutionary relations; and (iii) r-scan statistics that can be applied to the analysis of spacings of sequence markers.

Carbohydr Res, 1992 Jul 2, 231, 39 - 50
Determination of the structure of the capsular antigen of Escherichia coli O8:K46:H30, using FABMS and 2D-NMR spectroscopy; Dutton GG et al.; The structure of the capsular antigen from Escherichia coli O8:K46:H30 was elucidated by methylation analysis and 1D and 2D 1H- and 13C-NMR spectroscopy, and by methylation analysis, 1D- and 2D-NMR spectroscopy, and FABMS of the oligosaccharide-alditol obtained after dephosphorylation of the polymer with aqueous hydrofluoric acid . The capsular polymer is of the teichoic acid type and has the following repeating unit . {Formula: see text}

Br J Surg, 1992 Jul, 79(7), 648 - 52
Effect of biliary decompression on reticuloendothelial function in jaundiced rats; Ding JW et al.; The recovery of reticuloendothelial system (RES) function following decompression of obstructive jaundice was studied using a rat model with bile duct ligation and side-to-side choledochoduodenostomy . Histopathological changes in the liver were still present 5 weeks after relief of jaundice, while results of liver function tests had returned to normal . RES function evaluated by the blood clearance and organ uptake of radiolabelled Escherichia coli using a corrected phagocytic index gradually returned to normal following biliary decompression . The severely impaired RES activity noted 1 week after operation may explain the increased incidence of sepsis and renal insufficiency in the early period after biliary surgery in jaundiced patients.

J Intern Med, 1992 Jul, 232(1), 77 - 80
Bilateral emphysematous pyelonephritis resolving to medical therapy; Nagappan R et al.; We describe a case of bilateral emphysematous pyelonephritis, in a diabetic female, that responded to medical therapy alone . Her complete improvement is documented radiologically . Emphysematous pyelonephritis, as a cause of serious infection in diabetic patients, is briefly reviewed.

Biochem J, 1992 Jul 1, 285 ( Pt 1), 193 - 9
A recombinant human 'mini'-hexokinase is catalytically active and regulated by hexose 6-phosphates; Magnani M et al.; Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion . According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule . We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917 . This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c . The overexpressed 'mini'-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme . These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.

Biochem J, 1992 Jul 1, 285 ( Pt 1), 187 - 92
Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2; Miles CS et al.; The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine . The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods . The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme . For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect . However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups . In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme . Decreases in kinetic-isotope effects seen with {2-2H}lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step . Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer . We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

Biochem J, 1992 Jul 1, 285 ( Pt 1), 173 - 80
Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide; Hayes JD et al.; Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide . A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated . Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358 . The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously {McLellan, Kerr, Cronshaw & Hayes (1991) Biochem . J . 276, 461-469} . A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed . The murine GST expressed in E . coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide . This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver . However, the GST synthesized in E . coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c . Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E . coli, whereas the enzyme in mouse liver possesses a blocked N-terminus . Although sequencing showed that the purified Yc subunit from E . coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc . Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to the constitutively expressed rat liver Yc1 subunit . The significance of the fact that both mouse Yc and rat Yc2 exhibit high catalytic activity towards AFB1-8,9-epoxide, whereas rat Yc1 possesses little activity towards this compound, is discussed in terms of structure/function.

Appl Environ Microbiol, 1992 Jul, 58(7), 2266 - 70
Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms; Byrd JJ et al.; The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E . coli in marine and estuarine water . E . coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance . The concentration of pBR322 DNA remained stable in E . coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days . E . coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days . Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration . Cloning of DNA into the plasmid pUC8 did not alter the ability of E . coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm . We conclude that E . coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.

Mol Pharmacol, 1992 Jul, 42(1), 44 - 56
Molecular neurotoxicology of trimethyltin: identification of stannin, a novel protein expressed in trimethyltin-sensitive cells; Toggas SM et al.; The molecular basis of selective vulnerability of specific neuronal populations to neurotoxicants remains a key focus in neurotoxicology . Trimethyltin (TMT) selectively damages neurons in rodent and human central nervous system after a single exposure . By coupling subtractive hybridization with molecular cloning techniques, we isolated a cDNA specifically localized in TMT-sensitive cells . This 2.9-kilobase cDNA encodes a putative 10-kDa peptide of 88 amino acids, termed "stannin." In immunocytochemical experiments, antisera raised against the amino terminus of stannin exhibited strong immunoreactivity in TMT-sensitive neurons in the hippocampus and entorhinal cortex, areas previously identified by in situ hybridization . Northern blot and in situ hybridization experiments detected a 3.0-kilobase stannin mRNA in brain, spleen, and kidney; expression occurred as early as embryonic day 15 in rat brain and thymus . In situ hybridization in human hippocampus demonstrated a stannin mRNA in pyramidal and dentate gyrus neurons . High stringency Southern blot analysis of genomic DNA identified stannin homologs in rabbit, Drosophila, and human . These findings indicate that stannin is present in TMT-sensitive cells and may play a role in the selective toxicity of organotin compounds.

Neuron, 1992 Jul, 9(1), 15 - 26
Generation and migration of cells in the developing striatum; Halliday AL et al.; The development of the rat striatum was investigated using a combination of two histochemically distinguishable retrovirus vectors . Using this method, it was possible to identify clonal boundaries within the embryonic striatum and thus determine patterns of proliferation, migration, and some lineal relationships . Several novel aspects of striatal histogenesis were discovered . Striatal progenitor cells do not exhibit a stem cell pattern of division between embryonic day 15 (E15) and E19; a progenitor-progeny relationship appears to exist for ventricular zone and subventricular zone (SVZ) cells; striatal progenitors produce a variety of clone types; some SVZ cells migrate radially, and some migrate tangentially within the SVZ; and radial glia and presumptive neurons can occur in the same clone.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 6202 - 6
pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys; O'Keefe DO et al.; To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli . Cloned DT secreted to the periplasm of E . coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity) . Mutant forms of DT with reduced lethality for E . coli were selected by plating the bacteria under acidic conditions . CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells . We traced the altered cytotoxicity of CRM503 to a Glu-349----Lys mutation (E349K), one of three point mutations, within the B fragment . The E349K mutation alone inhibited cytotoxicity and membrane translocation in mammalian cells and lethality for E . coli but did not affect enzymic activity or receptor binding . The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic alpha-helices of the translocation domain . Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer . The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT . These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 6129 - 33
Reconstruction of a Streptomyces linear replicon from separately cloned DNA fragments: existence of a cryptic origin of circular replication within the linear plasmid; Shiffman D et al.; We report here the reconstruction of a functional linear replicon, the 12-kilobase Streptomyces clavuligerus plasmid pSCL, from separate DNA fragments cloned in Escherichia coli on the pUC19 plasmid . Protein-free DNA molecules containing the full-length pSCL sequence, an internally inserted thiostrepton-resistance gene, and adventitious nucleotides external to the pSCL termini were introduced into Streptomyces lividans, where the synthesis and functional attachment of replication proteins occurred and pSCL was established as an extrachromosomal linear replicon . Transformation of S . lividans with uncut supercoilded pUC19/pSCL DNA from E . coli or with a circularized 8-kilobase internal fragment of pSCL yielded circular replicons, indicating the existence of a cryptic origin of circular replication within the linear plasmid . Insertion mutations at sites that prevented the replication of pSCL linear plasmids also interfered with its replication in the circular mode.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 6114 - 8
Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase; Almassy RJ et al.; The three-dimensional structure of phosphoribosylglycinamide formyltransferase (10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2) has been solved both as an apoenzyme at 2.8-A resolution and as a ternary complex with the substrate glycinamide ribonucleotide and a folate inhibitor at 2.5-A resolution . The structure is a modified doubly wound alpha/beta sheet with flexibility in the active site, including a disordered loop in the apo structure, which is ordered in the ternary complex structure . This enzyme is a target for anti-cancer therapy and now for structure-based drug design.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5912 - 6
Nonconservative recombination in Escherichia coli; Takahashi NK et al.; Homologous recombination between two duplex DNA molecules might result in two duplex DNA molecules (conservative) or, alternatively, it might result in only one recombinant duplex DNA molecule (nonconservative) . Here we present evidence that the mode of homologous recombination is nonconservative in an Escherichia coli strain with an active RecF pathway (a recBC sbcBC mutant) . We employed plasmid substrates that enable us to recover both recombination products . These plasmids carry two mutant alleles of neo gene in direct orientation, two drug-resistance marker genes, and two compatible replication origins . After their transfer to the cells followed by immediate selection for the recombination to neo+, we could recover only one recombination product . A double-strand break at the region of homology increased this nonconservative recombination . If a nonconservative exchange should leave an end, this end may stimulate another exchange . Such "successive half crossing-over events" can explain several recombination-related phenomena in E . coli, including the origin of plasmid linear multimers and of transcribable, nonreplicated recombination products, and also in yeast and mammalian cells.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5892 - 6
Fumarase C, the stable fumarase of Escherichia coli, is controlled by the soxRS regulon; Liochev SI et al.; Fumarase C was strongly induced by paraquat in a parental strain of Escherichia coli but was not induced in a strain lacking the soxRS response . Moreover, a strain that constitutively expresses the soxRS regulon contained more fumarase C than did the parental strain . The Mn-containing superoxide dismutase and glucose-6-phosphate dehydrogenase, members of the soxRS regulon, were similarly induced by paraquat . Mutational defects in glucose-6-phosphate dehydrogenase increased the induction of fumarase C by paraquat . For Mn-containing superoxide dismutase, responsiveness to paraquat was also enhanced in the glucose-6-phosphate dehydrogenase-defective strains . Overproduction of the Mn-containing superoxide dismutase, elicited by isopropyl beta-D-thiogalactoside in a tac-sodA fusion strain, did not diminish induction of fumarase C or of glucose-6-phosphate dehydrogenase by paraquat, and induction of these enzymes was more sensitive to paraquat when the cells were growing on succinate rather than on LB medium . These results indicate that fumarase C is a member of the soxRS regulon and that this regulon does not respond to changes in O2- concentration but perhaps does respond to some consequence of a decrease in the ratio of NADPH to NADP+.

Mol Microbiol, 1992 Jul, 6(13), 1841 - 51
Expression of the Escherichia coli dam gene; Lobner-Olesen A et al.; The Escherichia coli dam gene and upstream sequences were cloned from the Kohara phage 4D4 . Five promoters were found to contribute to dam gene transcription . P1 and P2 (the major promoter) were situated approximately 3.5 kb upstream of the structural gene, P3 was within the aroB gene, P4 was within the urf74.3 gene, and P5 was in the urf74.3-dam intergenic region . The nucleotide sequence of 2280 bp of DNA containing P1 and P2 was determined and shown to have the potential to encode a protein of approximately 16 kDa between P1, P2 and the aroB gene . This 16 kDa open reading frame has been identified as aroK, the gene for shikimic acid kinase I . Thus the dam gene is part of an operon containing aroK, aroB, urf74.3, and dam . The transcriptional start points of the promoters were determined . A comparison of their nucleotide sequences suggested that P1-P4 were all recognized by the sigma 70 subunit of the RNA polymerase.

Mol Microbiol, 1992 Jul, 6(13), 1777 - 84
Phosphotransfer signal transduction between two regulatory factors involved in the osmoregulated kdp operon in Escherichia coli; Nakashima K et al.; The proteins KdpD and KdpE are regulatory factors critically involved in the osmotic regulation of the kdpABC operon that is responsible for a high-affinity transport system in Escherichia coli . In this study, we obtained biochemical evidence supporting the view that the KdpD protein is a sensory protein kinase that exhibits autophosphorylation and KdpE-phosphotransfer characteristics . During the course of such studies we established a procedure for purifying the KdpE protein in large quantities . We also developed a procedure for preparing cytoplasmic membrane enriched with the KdpD protein that exhibits in vitro ability with regard to phosphorylation of KdpE protein.

Mol Microbiol, 1992 Jul, 6(13), 1769 - 76
Clarification of the structural and functional features of the osmoregulated kdp operon of Escherichia coli; Sugiura A et al.; Expression of the Escherichia coli kdpABC operon, which is responsible for a high-affinity potassium-uptake system, is regulated in response to a change in the medium osmolarity . In this study, we clarified the structure and function of the kdpABC promoter including its regulatory sequence at the molecular level . The canonical -35 and -10 regions determined for the promoter were not fully functional, i.e . in addition to them, a cis-acting sequence located upstream of the -35 region was essential for full activation of the promoter . This upstream sequence was demonstrated to be the target site for the trans-acting activator, KdpE.

Mol Biol Evol, 1992 Jul, 9(4), 744 - 52
Sequencing errors and molecular evolutionary analysis; Clark AG et al.; Heuristic approaches were used to quantify the influence that sequencing errors have on estimates of nucleotide diversity, substitution rate, and the construction of genealogies . Error rates of less than 1 nucleotide/kb probably have little affect on conclusions about the evolutionary history of highly polymorphic organisms such as Drosophila and Escherichia coli, but organisms with very low nucleotide diversity, such as humans, require greater sequencing accuracy . A scan of GenBank for corrections of previous errors reveals that sequencing errors are highly nonrandom.

Mol Biol Evol, 1992 Jul, 9(4), 654 - 65
Molecular population genetics of Escherichia coli: DNA sequence diversity at the celC, crr, and gutB loci of natural isolates; Hall BG et al.; The DNA sequences of three genes--celC, crr, and gutB--have been determined for each of 11 or 12 natural isolates of Escherichia coli from the ECOR collection . These genes encode the phosphoenolpyruvate-dependent phosphotransferase-system enzyme III proteins specific for beta-glucoside sugars (celC), glucose (crr), and glucitol (gutB), respectively . There is little evidence of recombination at or among these loci; among these strains, relationships inferred from each gene are largely consistent with each other and with the relationship inferred from multilocus enzyme electrophoresis . DNA sequence diversity is similar for all three genes, particularly when silent (synonymous) sites only are considered . This is surprising because there is much stronger codon usage bias at crr than at celC or gutB . The extent of divergence in the protein sequences encoded by these three genes varies considerably . The constitutively expressed glucose-specific enzyme is completely conserved . It is surprising that the inducible glucitol-specific enzyme, which is functional, is more variable than the cellobiose-specific enzyme, which is cryptic; the latter might be expected to be under less (if any) purifying selection.

J Clin Microbiol, 1992 Jul, 30(7), 1896 - 8
Molecular epidemiology unravels the complexity of neonatal Escherichia coli acquisition in twins; Bingen EH et al.; Combined analysis of restriction fragment length polymorphism of regions of genes coding for rRNA (ribotyping) and esterase electrophoretic typing was used to document neonatal acquisition of Escherichia coli in twins . Our study shows vertical mother-to-infant transmission of one strain of E . coli to one twin and the development of neonatal septicemia with a distinct nonvirulent carboxylesterase type B1 E . coli strain for the other twin.

J Clin Microbiol, 1992 Jul, 30(7), 1823 - 8
Use of nonradioactive DNA hybridization for identification of enterotoxigenic Escherichia coli harboring genes for colonization factor antigen I, coli surface antigen 4, or putative colonization factor O166; Sommerfelt H et al.; We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166) . In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166 . A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria . In the rapid NCHA, densitometry verified the visual discrimination between four groups of E . coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E . coli strains that lack such genes . As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.

J Clin Microbiol, 1992 Jul, 30(7), 1801 - 6
Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome; Brian MJ et al.; Two pairs of oligonucleotide primers were designed to amplify fragments of the genes for Shiga-like toxin I (SLT-I) and SLT-II in a single reaction . A 370-bp segment and a 283-bp segment were amplified for SLT-I and SLT-II, respectively . The specificities of the polymerase chain reaction (PCR) amplification products were confirmed by using radioactively labeled oligonucleotide probes . SLT sequences were amplified from DNA isolated from 13 previously characterized enterohemorrhagic Escherichia coli (EHEC) strains . No amplification product was produced by using DNA from 20 non-EHEC strains . As little as one bacterial genome was detectable . PCR was then applied to DNA isolated directly from stool samples . We had to remove inhibitors of PCR that were present in lysates prepared from stool samples before amplification was achieved . First, we evaluated the sensitivity of PCR for the detection of known numbers of EHEC added to normal stools . Second, three children with SLT in their stools were shown to have SLT sequences in their stools by PCR . Two of these children had hemolytic-uremic syndrome, and a third child was asymptomatic . Stool specimens collected from another 26 asymptomatic children were negative by PCR for SLT sequences . PCR can be used to diagnose EHEC infections without prior culture of stool specimens.

Gene, 1992 Jul 1, 116(1), 75 - 80
A genetic system to study the in vivo role of transcriptional regulators in Escherichia coli; Perez-Martin J et al.; A genetic system for studying in vivo the interactions between a transcriptional regulatory protein and its target DNA has been developed for Escherichia coli . It is composed of two compatible plasmids: one high-copy-number promoter-probe vector, and one low-copy-number vector in which the gene encoding the desired protein is cloned under the control of an inducible promoter . The system was successfully tested for its specificity and for dosage analysis by using a combination of the plasmid pLS1-encoded RepA repressor and its target DNA.

Gene, 1992 Jul 1, 116(1), 7 - 12
Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi; York D et al.; The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology . We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA . In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected . We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough . Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation . Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene . The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo . We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight . Thus, at least three options exist in the translation of the wt pir mRNA . Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38 . Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).

Gene, 1992 Jul 1, 116(1), 59 - 67
Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase: use of pyrG for homologous transformation; Benito EP et al.; A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene of Mucor circinelloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe . The complete nucleotide sequence of the M . circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis . The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P . blakesleeanus . Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart . The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes . Northern-blot hybridization revealed the pyrG transcript to be approx . 1.0 kb . The M . circinelloides pyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coli when inserted between bacterial expression signals . Additionally, the genomic clone complemented the M . circinelloides pyrG4 mutation . When an M . circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per micrograms DNA and per 10(6) viable protoplasts.

Gene, 1992 Jul 1, 116(1), 13 - 20
A fusion plasmid for the synthesis of lipopeptide-antigen chimeras in Escherichia coli; Rioux CR et al.; Lipopeptides are potential vaccine candidates with a built-in adjuvant property . To circumvent the present chemical route of synthesis for lipopeptide-antigen conjugates, the lipoprotein property of the pColE2-P9-encoded lysis protein, CelB, was used to create the bacterial fusion plasmid, pKLY3, to produce lipopeptide-antigen chimeras in Escherichia coli . Plasmid pKLY3 is a derivative of pKK233-2 with the origin of replication of the single-stranded DNA phage, fl . Under control of the promoter, ptrc, is the 5' end of the celB gene coding for a lipoprotein signal peptide and the first five amino acids (aa) (CQANY) of the mature lysis protein . As model systems for the synthesis of small and large lipopeptide-antigens, DNA sequences coding for the P2 peptide and E . coli alkaline phosphatase (PhoA) were fused in frame to the region of celB coding for a lipoprotein signal peptide and CQANY . P2 is a 12-aa peptide including a tyrosine phosphorylation site of the epidermal growth factor receptor (EGF-R) . Inducible expression of stable lipohexapeptide CQANYV, lipo-CQANY-P2, and lipo-CQANYA-PhoA, was demonstrated . Similar expression was obtained for lipo-CIEGR-P2 and lipo-CIEGRA-PhoA in which IEGR is a cleavage recognition site for the blood coagulation factor, Xa . Like QANY, IEGR is predicted to form a beta-turn structure . The presence of a lipid moiety on the products was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin . The lipid-modified peptides were also identified by incorporation of radioactive tyrosine, and the nature of the P2 peptide was verified immunologically.(ABSTRACT TRUNCATED AT 250 WORDS)

Genes Dev, 1992 Jul, 6(7), 1165 - 72
DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli; Wild J et al.; In Escherichia coli secreted proteins must be maintained in an export-competent state before translocation across the cytoplasmic membrane . This function is carried out by a group of proteins called chaperones . SecB is the major chaperone that interacts with precursor proteins before their secretion . We report results indicating that the DnaK and DnaJ heat shock proteins are also involved in the export of several proteins, most likely by acting as their chaperones . Translocation of alkaline phosphatase, a SecB-independent protein, was inhibited in dnaK- and dnaJ- mutant strains, suggesting that export of this protein probably involves DnaK and DnaJ . In addition, DnaK and DnaJ play a critical role in strains lacking SecB . They are required both for viability and for the residual processing of the SecB-dependent proteins LamB and maltose-binding protein (MBP) seen in secB null strains . Furthermore, overproduction of DnaK and DnaJ permits strains lacking SecB to grow in rich medium and accelerates the processing of LamB and MBP . These results suggest that under conditions where SecB becomes limiting, DnaK and DnaJ probably substitute for SecB and facilitate protein export . This provides the cell with a mechanism to overcome a temporary imbalance in the secretion process caused by an abrupt expansion in the pool of precursor proteins.

Eur J Biochem, 1992 Jul 1, 207(1), 207 - 13
Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins . Analysis of rap and ras proteins in membranes from mammalian cells; Klinz FJ et al.; Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins . Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels . Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells . Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts . In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated . By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1 . An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells . In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.

Eur J Biochem, 1992 Jul 1, 207(1), 109 - 16
Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5; Peyronneau MA et al.; Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator {Renaud, J . P., Cullin, C., Pompon, D., Beaune, P . and Mansuy, D . (1990) Eur . J . Biochem . 194, 889-896} . The use of an improved expression vector {Urban, P., Cullin, C . and Pompon, D . (1990) Biochimie 72, 463-472} increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l . The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes . 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5 . Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5 . This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity . It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable . Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates . The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex . A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes . In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1992 Jul, 11(7), 2685 - 93
Unusual stability of recombination intermediates made by Escherichia coli RecA protein; Muller B et al.; The structure and stability of recombination intermediates made by RecA protein have been investigated following deproteinization . The intermediates consist of two duplex DNA molecules connected by a junction, as visualized by electron microscopy . Although we expected the structures to be highly unstable due to branch migration of the junction, this was not the case . Instead, we found that the intermediates were stable at 37 degrees C . At 56 degrees C, greater than 60% of the intermediates remained after 6 h of incubation . Only at higher temperatures was significant branch migration observed . This unexpected stability suggests that the formation of extensive lengths of heteroduplex DNA in Escherichia coli is likely to require the continued action of proteins, and does not occur via spontaneous branch migration . We show that heteroduplex DNA may be formed in vitro by ATP-dependent strand exchange catalysed by RecA protein or by the RuvA and RuvB proteins of E . coli.

EMBO J, 1992 Jul, 11(7), 2391 - 7
Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator; Mistou MY et al.; The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2 . These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS . The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21 . Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain . No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21 . Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21 . No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP) . This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors . Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.

Am Rev Respir Dis, 1992 Jul, 146(1), 190 - 5
Late effects of endotoxin on the accumulation and function of monocytes in rabbit lungs; Ohgami M et al.; Recent studies from our laboratory show that the lung contains a marginated pool of monocytes . The present study was designed to investigate monocyte accumulation in this pool 4 to 28 h after a single dose of endotoxin when the endotoxin had disappeared from the circulation . This was accomplished by administering a single intravenous dose of endotoxin (Escherichia coli, 50 micrograms/rabbit) to unanesthetized animals (n = 6) and saline to controls (n = 5) at time 0 . Four hours after this dose of endotoxin, 111In-monocytes (93.5% pure) isolated from donors were injected intravenously, and, at 27 h, the rabbits were anesthetized and colloidal carbon (CC, 1 ml/kg body weight) was injected intraarterially to provide a phagocytic stimulus . The animals were sacrificed at 28 h, and the lungs were fixed in situ with glutaraldehyde . The data show that lungs from the endotoxin-treated rabbits contained 4.8 times more 111In-monocytes than controls, that 92% of these radiolabeled monocytes were in the alveolar capillaries, and that 72% of these labeled cells had phagocytosed CC . The histologic studies of unlabeled cells confirmed that this endotoxin treatment caused a 3-5-fold increase in unlabeled mononuclear cells and neutrophils (PMN) in the microvasculature and that many of the unlabeled monocytes in the endotoxin-treated group had also phagocytosed colloidal carbon . The behavior of the donor monocytes injected after the endotoxin had time to disappear from the circulation suggests that they accumulate in the lung in response to the indirect effects of endotoxin on endothelial cells.

J Pharmacol Exp Ther, 1992 Jul, 262(1), 439 - 44
Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli; Tanaka H et al.; The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF) . Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF . The effects of 5637- and recombinant hG-CSF administered via i.v . injection to rats showed similar response patterns of neutrophil counts in peripheral blood . From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities . The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay . After i.v . administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially . Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form . After s.c . administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.

J Bacteriol, 1992 Jul, 174(14), 4850 - 2
Interallelic complementation of dnaE(Ts) mutations; Bryan SK et al.; Some Escherichia coli dnaE(Ts) alleles will functionally complement in trans . The complementation is not due to copy number and is compatible with dimeric interaction.

J Bacteriol, 1992 Jul, 174(14), 4842 - 6
The replication initiator operon of promiscuous plasmid RK2 encodes a gene that complements an Escherichia coli mutant defective in single-stranded DNA-binding protein; Jovanovic OS et al.; The amino acid sequence of the 13-kDa polypeptide (P116) encoded by the first gene of the trfA operon of IncP plasmid RK2 shows significant similarity to several known single-stranded DNA-binding proteins . We found that unregulated expression of this gene from its natural promoter (trfAp) or induced expression from a strong heterologous promoter (trcp) was sufficient to complement the temperature-sensitive growth phenotype of an Escherichia coli ssb-1 mutant . The RK2 ssb gene is the first example of a plasmid single-stranded DNA-binding protein-encoding gene that is coregulated with replication functions, indicating a possible role in plasmid replication.

J Bacteriol, 1992 Jul, 174(14), 4811 - 9
Elasticity of the sacculus of Escherichia coli; Koch AL et al.; Preparations of purified peptidoglycan of Escherichia coli (i.e., sacculi) were studied by low-angle laser light scattering . Control experiments and theoretical calculations based on the Rayleigh-Gans theory showed that the mean sacculus surface area could be accurately inferred from measurements with our apparatus by using computer routines developed previously . Large changes in the mean saccular surface area resulted from alterations in the stress caused by varying the net charge on the sacculi . The net charge was affected by altering the suspending medium pH, causing carboxyl and amino groups in the peptidoglycan to gain or lose protons, or by acetylation or succinylation of the amino groups . A preponderance of either plus or minus charges caused an expansion of the mean sacculus surface area . The largest increase in area probably represents the elastic limit of the peptidoglycan and was 300% above the area of isoionic sacculi . This degree of expansion is consistent with possible conformations of the intact peptidoglycan structure without necessitating rupture of the wall fabric . Our findings concerning saccular elasticity provide support for the surface stress theory . It provides a mechanism so that bacteria can grow and divide while maintaining turgor pressure, without the necessity of having and using proteins to do the mechanical work.

J Bacteriol, 1992 Jul, 174(14), 4777 - 82
Identification of a novel promoter in the replication control region of plasmid R6K; Mukerji P et al.; A novel source of transcription has been detected in the replication region of plasmid R6K by using fusions involving the galK reporter gene . The -35 and -10 consensus RNA polymerase binding sites were identified in the region overlapping the binding sites for the R6K-encoded replication protein pi . Transcription from this promoter, designated P2, is repressed in vivo by pi-protein levels that are inhibitory for replication . Promoter-down mutations in P2 induced in vitro by bisulfite mutagenesis result in a reduced copy number of a beta-replicon but not of a gamma-replicon . Implications of the role of P2 in R6K replication are discussed.

J Bacteriol, 1992 Jul, 174(13), 4513 - 6
Genetic and morphological characterization of an Escherichia coli chromosome segregation mutant; Stewart PS et al.; The temperature-sensitive nucleoid segregation mutant of Escherichia coli, PAT32, formerly described as a parA mutant, has been shown to carry a mutation near 66 min on the genetic map . Fine mapping with phages from the collection of Kohara et al . is consistent with its being a parC allele . Observation by fluorescence microscopy revealed the formation, at a nonpermissive temperature, of filaments containing one or two large nucleoids and of normal-size anucleate cells . There was also a significant loss of viability.

J Bacteriol, 1992 Jul, 174(13), 4457 - 62
Replication of prophage P1 is cell-cycle specific; Keasling JD et al.; P1 prophage replication during the Escherichia coli division cycle has been analyzed by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive prophage DNA . P1 prophage replicates during a restricted portion of the bacterial division cycle, like the minichromosome, but at a time during the division cycle different than the time at which the minichromosome replicates in the same cell . A high-copy mini-R6K plasmid present in the same cell replicates throughout the division cycle . Over a wide range of growth rates, the P1 prophage replicates approximately one-half generation after the minichromosome replicates . Thus, the mechanisms underlying P1 replication are similar to those for the F plasmid and the chromosome . Replication occurs when some property related to cell size or cell mass reaches a constant value per origin.

J Bacteriol, 1992 Jul, 174(13), 4450 - 6
Multiple mutant of Escherichia coli synthesizing virtually thymineless DNA during limited growth; el-Hajj HH et al.; The dut gene of Escherichia coli encodes deoxyuridine triphosphatase, an enzyme that prevents the incorporation of dUTP into DNA and that is needed in the de novo biosynthesis of thymidylate . We produced a conditionally lethal dut(Ts) mutation and isolated a phenotypic revertant that had a mutation in an unknown gene tentatively designated dus (for dut suppressor) . The dus mutation restored the ability of the dut mutant to grow at 42 degrees C without restoring its enzymatic activity or thymidylate independence . A strain was constructed bearing, in addition to these mutations, ones affecting the following genes and their corresponding products: ung, which produces uracil-DNA N-glycosylase, a repair enzyme that removes uracil from DNA; deoA, which produces thymidine (deoxyuridine) phosphorylase, which would degrade exogenous deoxyuridine; and thyA, which produces thymidylate synthase . When grown at 42 degrees C in minimal medium containing deoxyuridine, the multiple mutant displayed a 93 to 96% substitution of uracil for thymine in new DNA . Growth stopped after the cellular DNA had increased 1.6- to 1.9-fold and the cell mass had increased 1.7- to 2.7-fold, suggesting a general failure of macromolecular biosynthesis . DNA hybridization confirmed that the uracil-containing DNA was chromosomal and that new rounds of initiation must have occurred during its synthesis.

J Bacteriol, 1992 Jul, 174(13), 4294 - 301
Disruption of the gene for Met-tRNA(fMet) formyltransferase severely impairs growth of Escherichia coli; Guillon JM et al.; In bacteria, as well as in chloroplasts and mitochondria, the free amino group of the methionylated initiator tRNA(fMet) is specifically modified by the addition of a formyl group . The importance of this modification remains unclear . With the availability of pure Escherichia coli 10-formyltetrahydrofolate:L-methionyl-tRNA(fMet) N-formyltransferase, the enzyme catalyzing Met-tRNA(fMet) formylation, the corresponding fmt gene and its flanking regions were cloned and sequenced . The chromosomal fmt gene was disrupted, and strains modified in their formylation activity were constructed . A depletion of the cellular formylation activity was accompanied by a decrease in the growth rate of the bacteria . At 37 degrees C, in a rich medium, the absence of a functional fmt gene reduced the growth rate to 0.28 doubling per h, from 2.3 for the control strain . At 42 degrees C, the studied fmt mutant strain did not grow further.

J Bacteriol, 1992 Jul, 174(13), 4258 - 64
Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2; Colbeau A et al.; The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus . DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase . We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression . The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis . Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures . Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis.

J Bacteriol, 1992 Jul, 174(13), 4205 - 11
The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth; Tsuchimoto S et al.; We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame . The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein . This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein . We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ . The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells . However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells . This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function . We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s) . In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above . Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.

Oncogene, 1992 Jul, 7(7), 1361 - 9
Phosphorylation of the p53 tumour-suppressor protein at three N-terminal sites by a novel casein kinase I-like enzyme; Milne DM et al.; Wild-type mouse p53, expressed in Escherichia coli, was phosphorylated by highly purified casein kinase I (CKI) from rabbit muscle . The major site of phosphorylation in the p53 was identified as serine 6, which is known to be phosphorylated in vivo . Serines 4 and 9 were also phosphorylated . To determine whether CKI is likely to be a physiological p53 kinase, SV3T3 cell lysates were fractionated on a Mono Q column and assayed for p53 kinase and casein kinase activities . Four p53 kinase activities were detected, one of which co-purified with CKI activity . This p53 kinase (designated PK270) further co-purified with CKI on sucrose gradients and had a native molecular weight, like CKI, in the range of 35,000-45,000 . However, PK270 was separated from the bulk of CKI activity on a phosvitin-Sepharose affinity column, and was therefore likely to be a CKI-related kinase . In support of these conclusions, phosphorylation of p53, by both CKI and PK270, was inhibited by a peptide corresponding to a consensus CKI target sequence, but not by a non-specific peptide . Moreover, phosphopeptide analyses of p53 phosphorylated by CKI or by PK270 gave similar results, indicating that these two kinases phosphorylate the same sites in p53.

Mol Cell Biol, 1992 Jul, 12(7), 3288 - 96
Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation; Feldheim D et al.; SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J . A . Rothblatt, R . J . Deshaies, S . L . Sanders, G . Daum, and R . Schekman, J . Cell Biol . 109:2641-2652, 1989) . Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network . Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein . A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane . The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol . Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein . This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen . By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E . coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus . Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity . Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus . A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

Metabolism, 1992 Jul, 41(7), 698 - 705
Influence of omega-3 fatty acids on splanchnic blood flow and lactate metabolism in an endotoxemic rat model; Pscheidl EM et al.; Alteration in regional blood flow is important in the pathogenesis of organ failure during endotoxemia and sepsis . In particular, intestinal ischemia is thought to enhance the translocation of bacteria into the systemic circulation . We used radioactive microspheres to measure the influence of two intravenous (IV) dietary fats (vegetable oil containing high levels of omega-6 fatty acids, and fish oil containing high levels of omega-3 fatty acids) on regional blood flow during low-dose Escherichia coli endotoxin infusion (0.1 mg/100 g body weight {BW}) in a rat model . Despite absence of changes in the cardiac output, blood flow rates to the small and large intestines, stomach, and pancreas, and also to the skin and skeletal muscle were significantly reduced after 18 hours of endotoxin infusion in the rats fed standard vegetable oil . Short-term IV feeding during a period of 40 hours with an isonitrogenous, isocaloric nutrient solution containing fish oil as the only lipid source normalized intestinal perfusion and increased blood flow to the liver and spleen . Low-dose endotoxin infusion also resulted in significant increases in glucose, lactate, and pyruvate concentrations . In comparison to standard vegetable fat emulsion, fish oil significantly reduced these parameters . A second experiment was conducted to measure lactate kinetics . Based on the dilution of U-14C-lactate, fish oil feeding was associated with higher lactate clearance than standard vegetable oil feeding during the endotoxin infusion . We conclude that short-term IV feeding with fish oil improves intestinal perfusion and portal blood flow, improves glucose tolerance, and increases lactate clearance in a low-dose endotoxin rat model.

J Cell Biol, 1992 Jul, 118(1), 33 - 42
Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli; Fujishige A et al.; alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J . L., C . N . McGrath, K . R . Smith, and D . A . Agard . 1988 . Gene (Amst.) . 69: 237-244) . The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D . R . 1970 . Methods Enzymol . 19:599-613) . Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J . L., D . Frank, A . Fujishige, R . Bone, and D . A . Agard . 1989 . J . Bacteriol . 171:1320-1325) or in trans (Silen, J . L., and D . A . Agard . 1989 . Nature (Lond.) . 341:462-464) for the proper folding and extracellular accumulation of the enzyme . The maturation process is temperature sensitive in E . coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J . L., D . Frank, A . Fujishige, R . Bone, and D . A . Agard . 1989 . J . Bacteriol . 171:1320-1325) . Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E . coli outer membrane . The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures . When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane . However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium . Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful . Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.

Dig Dis Sci, 1992 Jul, 37(7), 1039 - 44
Evidence that gastric antisecretory action of lipopolysaccharide is not due to a toxic effect on gastric parietal cells; Uehara A et al.; We have recently found that bacterial lipopolysaccharide (LPS) or endotoxin at minute doses inhibits the secretion of gastric acid and pepsin in rats . The present study was performed to determine whether this antisecretory action of LPS was a reversible biological response or a result of the destruction of gastric parietal cells by endotoxin . The intraperitoneal injection of LPS into pylorus-ligated rats resulted in a dose-related (40-4000 ng/kg) decrease in gastric acid secretion, with maximal inhibition being observed at a dose of 4000 ng/kg . The stomach then was examined both macroscopically and microscopically for the presence or absence of mucosal lesions or damaged gastric parietal cells . No morphological changes in the gastric mucosal structure including parietal cells were observed even in the rats injected with 4000 ng/kg of LPS . Next, basal gastric acid output was compared in the rats that had received LPS (4000 ng/kg, intraperitoneal) or saline alone 24 hr before . There was no significant difference in gastric acid secretion between the saline- and LPS-pretreated groups, indicating that the secretory capacity of gastric parietal cells returned to the control level at 24 hr after the injection of a maximal antisecretory dose of LPS . These results clearly suggest that the LPS-induced inhibition of gastric secretion results not from its toxic or destructive effect on the gastric secretory mechanism but from its reversible biological effect on gastric physiology.

Crit Care Med, 1992 Jul, 20(7), 1005 - 13
Pressure-flow relationships of the pulmonary circulation during endotoxin infusion in intact dogs; D'Orio V et al.; BACKGROUND AND METHODS: We aimed to characterize the effects of an endotoxin insult (Escherichia coli 0127:B8) on the relationships between pulmonary vascular pressure and flow in intact dogs . To achieve this goal, multipoint plots of total pressure gradient, arterial pressure gradient, and venous pressure gradient vs . flow were generated by graded inflation of a right atrial balloon, which was used to vary flow . The partitioning of the total pressure decrease across the pulmonary vasculature (total pressure gradient = pulmonary arterial pressure-pulmonary artery occlusion pressure {PAOP}) into gradients across pulmonary arterial (arterial pressure gradient = pulmonary arterial pressure--effective capillary pressure) and pulmonary venous (venous pressure gradient = effective capillary pressure--PAOP) regions was assessed by a waveform mathematical analysis of the pulmonary arterial pressure profile during arterial occlusion, with computation of both PAOP and effective pulmonary capillary pressures . Slopes and extrapolated pressure intercepts from linear regression fits to the pulmonary vascular pressure/flow plots were determined in seven dogs after a 2-hr endotoxic infusion interval and were compared with the corresponding values that characterized a similar group of sham-operated dogs . RESULTS: Under normal conditions, the extrapolated pressure intercept for pulmonary arterial pressure gradient was virtually 0 mm Hg; for total pulmonary arterial pressure gradient and pulmonary venous pressure gradient, the mean extrapolated pressure intercepts were substantially positive: 2.4 +/- 0.2 and 2.1 +/- 0.3 mm Hg, respectively . Endotoxin infusion at 0.25 micrograms/kg/min significantly increased the pressure intercepts from 2.4 to 8.7 and from 2.1 to 8.3 mm Hg of total pressure gradient and venous pressure gradient vs . flow, respectively . This infusion produced a minor, nonsignificant change in the intercept of arterial pressure gradient vs . flow, whereas it increased its slope significantly (p less than .05) from 0.036 to 0.081 mm Hg/mL/min/kg . CONCLUSIONS: These data suggest that endotoxin's effects on vascular resistance are exerted at two different loci such that these effects are additive . These endotoxin-induced effects consisted of increased vascular resistance of the arterial segment and appearance of a Starling resistor at the venous side of the pulmonary circulation, which acted as the relevant back-pressure to flow.

Cancer Res, 1992 Jul 1, 52(13), 3693 - 7
Oxidation of methylhydrazines to mutagenic methylating derivatives and inducers of the adaptive response of Escherichia coli to alkylation damage; Sedgwick B; The methylhydrazines, monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine, are known carcinogens but only weak mutagens in the Ames test . Chemical oxidation of these compounds by potassium ferricyanide greatly enhanced their mutagenicity to an Escherichia coli ada mutant and converted them into inducers of the adaptive response of E . coli to alkylation damage . Enzymatic oxidation of monomethylhydrazine by horseradish peroxidase-H2O2 also yielded products which induced the adaptive response . Thus, methylhydrazines can be oxidized to active DNA-methylating derivatives which generate methylphosphotriesters (the inducing signal of the adaptive response), O6-methylguanine and/or O4-methylthymine (the miscoding bases repaired by the Ada protein) in DNA . These observations support the suggestion that metabolic oxidation of methylhydrazines in mammalian systems may be required to generate the mutagenic/carcinogenic derivatives.

Cancer Res, 1992 Jul 1, 52(13), 3521 - 7
Inhibition of dihydroorotate dehydrogenase activity by brequinar sodium; Chen SF et al.; The novel anticancer drug candidate brequinar sodium (DuP 785, NSC 368390, 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline- carboxylic acid sodium salt) was shown previously to be an inhibitor of dihydroorotate dehydrogenase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway . Brequinar sodium inhibits the activity of this enzyme isolated from mammalian sources only but not those forms isolated from yeast or bacteria, which also use ubiquinone as the cofactor . Brequinar sodium also does not inhibit the activity of a soluble Zymobacterium oroticum dihydroorotate dehydrogenase which uses NAD+ as a cofactor . Brequinar sodium inhibits L1210 dihydroorotate dehydrogenase with mixed inhibition kinetics with respect to either the substrate (dihydroorotate) or the cofactor (ubiquinone Q6) with Ki' values in the 5-8 nM range . Our results suggest that brequinar sodium inhibits dihydroorotate dehydrogenase by binding to the enzyme at a unique site that is distinct from the dihydroorotate or the ubiquinone-binding site . This binding site appears to be unique to the mammalian enzyme, because brequinar sodium does not inhibit the yeast, Escherichia coli, or Z . oroticum forms of the enzyme.

Proc Soc Exp Biol Med, 1992 Jul, 200(3), 353 - 8
Impaired febrile response with age: role of thermogenesis in brown adipose tissue; Scarpace PJ et al.; We demonstrated previously that in Escherichia coli-infected rats, the heat necessary for the febrile response is a result of thermogenesis in brown adipose tissue (BAT) . To investigate whether senescent rats have an impaired febrile response to infection and whether such an impairment is a result of attenuated sympathetically activated thermogenesis in BAT, we assessed body temperature and the increase in mitochondrial guanosine 5'-diphosphate (GDP) binding sites in interscapular BAT in response to E . coli administration in young and senescent male F-344 rats . There was a significant delay of 2 hr in the onset of fever in the older animals . In addition, in senescent rats, the peak fever (1.0 +/- 0.1 delta degrees C vs 2.2 +/- 0.1) and the cumulative fever (383 +/- 43 delta degrees C.min vs 775 +/- 69) were significantly less than in the young rats (P less than 0.005) . Baseline levels of GDP binding were the same in young and old rats . In young rats, during the rising phase of the fever, E . coli infection resulted in a 50% increase in the density of GDP binding sites in BAT mitochondria . In contrast, there was no increase in GDP binding in the older rats following infection . The failure to increase GDP binding may be a result of a reduced ability to unmask reserve GDP binding sites . Alternatively, there may be fewer total GDP binding sites (masked and unmasked) in senescent rats and these sites may already be unmasked . Collectively, these data suggest that the impaired febrile response with age is due to reduced thermogenesis in BAT.

Proc Soc Exp Biol Med, 1992 Jul, 200(3), 338 - 42
Enhanced growth restriction of Legionella pneumophila in endotoxin-treated macrophages; Egawa K et al.; Macrophages from A/J mice are permissive for growth of Legionella pneumophila, an intracellular opportunistic pathogen that grows preferentially in macrophages . Macrophages from other mouse strains are highly resistant to growth of Legionella . In the present study, it was found that macrophages from A/J mice are readily activated by pretreatment with lipopolysaccharide (LPS), so that the cells do not permit Legionella to replicate in vitro, as occurs when untreated macrophages from A/J mice are cultured with these organisms for 48 hr . The augmentation of Legionella growth inhibition by LPS-activated macrophages from nonpermissive BDF1 mice also occurred . After in vitro infection, there was a 1000-fold increase in the number of Legionella in A/J macrophages and approximately a 10-fold increase in BDF1 macrophages, but LPS treatment of macrophages from either strain resulted in marked growth restrictions . This suppression was both dose dependent as well as dependent upon the time of addition of the LPS to the macrophages . Furthermore, the lipid A component of LPS was found to be as effective as the intact LPS in activating macrophages to inhibit the intracellular growth of Legionella . Further studies concerning the mechanisms involved are clearly warranted and in progress.

J Urol, 1992 Jul, 148(1), 173 - 8
Characterization of bovine bladder mucin fractions that inhibit Escherichia coli adherence to the mucin deficient rabbit bladder; Ruggieri MR et al.; We have previously shown that a dialyzed, lyophilized saline extract from bovine urothelium can restore the antiadherence activity of the rabbit bladder following mucin removal with 50% acetone . This report describes results of initial purification of antiadherence factor(s) from bovine bladder mucin and describes results of biochemical analysis in an attempt to elucidate possible mechanism of this antiadherence activity . Separation performed by gel filtration (Spectra gel AcA 34, 20-350 KD range) results in three fractions . Only the low molecular weight fraction had a statistically significant inhibitory effect on bacterial adherence to the mucin deficient rabbit bladder . After overnight dialysis against running deionized water and lyophilization, the crude extract contained 60% protein while gel filtration fractions 1-3 contained 35%, 90% and 15% by weight respectively . The first fraction (apparent high molecular weight, greater than 350 kD) did not appear to enter SDS polyacrylamide electrophoresis gels (SDS-PAGE, 3-12%) or agarose gels (0.5%) to any significant extent . In the fractions that displayed antiadherence activity (the crude extract, fractions 2 and 3) SDS-PAGE bands were seen corresponding to an apparent molecular weight of 78 kD in addition to bands co-migrating with bovine serum albumin (BSA) . BSA itself slightly increases bacterial adherence in this model . Most of the albumin of the crude extract was found in the second fraction (60%) . On the other hand most of the sulfate and sugar of the crude extract was found in the third, low molecular weight fraction . Since sulfated polysaccharides such as heparin and dextran sulfate are very effective antiadherence agents in this rabbit bladder model, it is conceivable that the sulfated sugar content of the third fraction is responsible for its antiadherence effect on the mucin deficient rabbit bladder.

Infect Immun, 1992 Jul, 60(7), 2920 - 5
Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein; Koopman MB et al.; A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S . hyodysenteriae periplasmic flagella . A single immunoreactive plaque was chosen for further analysis . The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella . DNA sequence analysis showed that the gene encodes a protein of 320 amino acids . The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins . We designated the cloned S . hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA . The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm . Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein . Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified . Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.

Infect Immun, 1992 Jul, 60(7), 2914 - 9
Interaction of Mycoplasma dispar with bovine alveolar macrophages; Almeida RA et al.; The capacity to avoid phagocytosis and the activation of bovine alveolar macrophages (BAM) by encapsulated Mycoplasma dispar or purified M . dispar capsule was investigated . Encapsulated and unencapsulated M . dispar were cocultured with BAM in the presence or absence of antisera prepared against unencapsulated M . dispar or purified capsule antiserum . Unopsonized mycoplasmas resisted phagocytosis, while only anti-capsule antibodies enhanced the phagocytosis of encapsulated mycoplasmas . BAM were cultured in the presence of purified M . dispar capsule or either live or heat-killed encapsulated or unencapsulated M . dispar . These BAM were then activated with Escherichia coli endotoxin or left without further activation . The supernatants of these cultures were assayed for tumor necrosis factor, interleukin 1, and glucose consumption as indicators of macrophage activation . Tumor necrosis factor and interleukin 1 were produced by BAM stimulated with unencapsulated M . dispar but not when encapsulated M . dispar or its purified capsule was used . Similarly, glucose consumption was increased in the presence of unencapsulated M . dispar, but not when BAM were cocultured with encapsulated M . dispar or purified capsule . When BAM were treated with purified capsule or encapsulated mycoplasmas, they could not be subsequently activated by endotoxin . These results indicate that encapsulated M . dispar or purified capsule exerts an inhibitory effect on the activity of BAM and prevents the activation of these cells.

Infect Immun, 1992 Jul, 60(7), 2641 - 7
In vivo effects of the antiglucocorticoid RU 486 on glucocorticoid and cytokine responses to Escherichia coli endotoxin; Hawes AS et al.; The endogenous adrenocortical response to sepsis is critical for host survival . The in vivo interactions among the endogenous glucocorticoid response, the induction of cytokines, and host survival during endotoxemia were explored in this study by use of the glucocorticoid receptor antagonist RU 486 . Male Lewis rats underwent sterile insertion of a right jugular venous catheter . After a 72-h recovery period, animals received a 50% lethal dose of Escherichia coli endotoxin (2.5 mg/kg) via the catheter after pretreatment for 30 min prior to lipopolysaccharide (LPS) treatment with (i) vehicle alone intravenously (i.v.) (-corticosterone {-Cort}/-RU 486/+LPS) (n = 10), (ii) the antiglucocorticoid RU 486 (10 mg/kg) i.v . (-Cort/+RU 486/+LPS) (n = 11), or (iii) RU 486 (10 mg/kg) i.v . in animals that had undergone subcutaneous implantation of a corticosterone pellet at the time of catheter insertion (+Cort/+RU 486/+LPS) (n = 10) . Except in animals receiving corticosterone pretreatment, baseline plasma corticosterone levels were low in all groups . Plasma corticosterone levels increased significantly (P less than 0.001) above the baseline following LPS administration . Animals in the -Cort/+RU 486/+LPS-treated group exhibited significantly increased mortality (P less than 0.001), with only 9% of the animals surviving at 72 h, as well as significantly increased plasma interleukin-6 levels, compared with animals receiving the vehicle alone (-Cort/-RU 486/+LPS), which showed 50% mortality . Pretreatment with corticosterone and RU 486 (+Cort/+RU 486/+LPS) significantly (P less than 0.001) reversed the mortality observed with RU 486 pretreatment alone (-Cort/+RU 486/+LPS), with 70% of the animals surviving at 72 h, and significantly attenuated the peak plasma tumor necrosis factor and interleukin-6 responses to LPS, compared with those in the animals treated with vehicle alone . These data demonstrate that the blockade of glucocorticoid binding by RU 486 increases LPS-induced mortality . The reversal of this effect by the induction of hypercorticosteronemia prior to RU 486 and LPS exposure (+Cort/+RU 486/+LPS) improves survival and is further associated with significant attenuation of cytokine production . Therefore, these data suggest that the protective effect of the endogenous glucocorticoid response to acute endotoxemia may result from the down-regulation of a potentially lethal cytokine response.

Infect Immun, 1992 Jul, 60(7), 2605 - 11
Antibody-secreting cells in human peripheral blood after oral immunization with an inactivated enterotoxigenic Escherichia coli vaccine; Wenneras C et al.; Vaccine antigen-specific antibody-secreting cell (ASC) responses in peripheral blood of healthy adult volunteers were studied after oral immunization with a prototype enterotoxigenic Escherichia coli (ETEC) vaccine by means of the enzyme-linked immunospot technique . Three doses of vaccine consisting of formalin-killed ETEC bacteria expressing fimbrial colonization factor antigens I and II (CFA/I and CFA/II) in combination with purified cholera toxin B subunit (CTB) were given 2 weeks apart . The ASC responses were detected 7 days after each immunization . Immunoglobulin A (IgA) was the predominant isotype produced by CFA/I- as well as CFA/II-specific ASCs . Moderate CFA/I- and CFA/II-specific IgM-secreting ASC (IgM-ASC) responses were also seen, whereas IgG-ASC responses to either of the CFAs were negligible . The ASC responses to CTB, on the other hand, comprised both IgA- and IgG-ASCs, with few if any specific IgM-ASCs . Almost 90% of the volunteers developed CFA-specific ASC responses after vaccination . Maximal CFA-specific ASC responses were usually observed after a single dose or two doses of vaccine . A third dose of vaccine did not result in increased but rather resulted in decreased magnitudes of CFA-specific ASC responses . Furthermore, it was found that CTB did not function as a mucosal adjuvant, since CFA-specific ASC responses were not enhanced by the simultaneous administration of CTB . These results suggest that two oral doses of ETEC vaccine induce a strong mucosal immune response, as reflected by the presence of large numbers of antigen-specific mucosal B cell immunoblasts in the blood.

Infect Immun, 1992 Jul, 60(7), 2593 - 7
Oral ingestion of egg yolk immunoglobulin from hens immunized with an enterotoxigenic Escherichia coli strain prevents diarrhea in rabbits challenged with the same strain; O'Farrelly C et al.; White Leghorn hens were immunized with enterotoxigenic Escherichia coli B16-4 with heat-labile enterotoxin and colonization factor antigen I in Freund's adjuvant . Specific antibodies were detected by an enzyme-linked immunosorbent assay in the serum after 8 days and in eggs after 10 days, with levels reaching peaks at 15 and 20 days after the first immunization, respectively . The protective effects of the egg yolk antibodies were tested in the rabbit reversible ileal tie model of diarrhea . Five control rabbits developed severe diarrhea within 72 h after inoculation with enterotoxigenic E . coli B16-4 . Oral ingestion of egg yolks from immunized hens for 4 days prior to inoculation protected five rabbits from diarrhea after challenge with the same strain of E . coli . The rabbits showed no adverse effects from the ingestion of the egg yolks . Four rabbits fed control eggs were also afforded some protection in that three rabbits developed mild diarrhea and one rabbit remained entirely well . In vitro experiments showed that immunoglobulin from egg yolks interfered with the binding of E . coli to purified small bowel mucins; immunoglobulin from immunized hens reduced binding more than immunoglobulin from nonimmunized hens . These findings indicate that eggs from hens immunized with appropriate antigens have potential as a useful source of passive immunity.

Infect Immun, 1992 Jul, 60(7), 2581 - 7
In vivo inhibition of lipopolysaccharide-induced lethality and tumor necrosis factor synthesis by Rhodobacter sphaeroides diphosphoryl lipid A is dependent on corticosterone induction; Zuckerman SH et al.; Diphosphoryl lipid A from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides (Rs-DPLA) has been demonstrated to block in mice and guinea pigs the increase in the serum tumor necrosis factor (TNF) response induced by highly purified deep rough chemotype LPS from Escherichia coli D31m4 (ReLPS) . The present study was designed to determine the role of corticosterone induction by Rs-DPLA and its effect on TNF regulation and survival in lethal endotoxin shock models and to evaluate the ability of Rs-DPLA to induce endotoxin tolerance . Administration of a 100-fold excess of Rs-DPLA 1 h prior to ReLPS administration inhibited the characteristic peak in serum TNF levels induced by LPS . Inhibition was apparent in normal and D-galactosamine (GalN)-sensitized mice and occurred at the pretranslational level, as splenic TNF and interleukin-1 beta mRNAs were present in lower amounts in LPS-stimulated mice pretreated with Rs-DPLA . Consistent with its effects in reducing serum TNF levels, Rs-DPLA pretreatment protected GalN-sensitized mice from a lethal ReLPS challenge . In contrast, Rs-DPLA did not inhibit the increase in the serum TNF response or protect against a lethal ReLPS challenge in parallel experiments with adrenalectomized (Adrex) mice, for which the 50% lethal dose of ReLPS was comparable to that for GalN-sensitized mice . Furthermore, Rs-DPLA appeared to prime Adrex animals and increase the magnitude of the serum TNF response to a suboptimal LPS stimulus . Priming by Rs-DPLA, however, was not observed in normal or GalN-sensitized mice . Although Rs-DPLA by itself was nontoxic and unable to elevate serum TNF levels in any of the models investigated, it did induce a significant increase in the serum corticosterone response and was capable of inducing endotoxin tolerance in normal mice . The inability of Rs-DPLA to protect Adrex mice from a lethal ReLPS stimulus or to inhibit the increase in the serum TNF response suggests that the protective effect of Rs-DPLA in normal or GalN-sensitized animals occurs through corticosterone induction . These results support the concept that endogenous glucocorticoids can modulate the endotoxic effects of LPS by inhibiting the synthesis of inflammatory cytokines.

Gastroenterology, 1992 Jul, 103(1), 282 - 8
Hemodynamic effects of endotoxin and platelet activating factor in cirrhotic rats; Kleber G et al.; Acute infections may influence the hemodynamic alterations of liver disease . Therefore, the hemodynamic effects of endotoxin (LPS E . coli 0111:B4) in conscious, normal, and cirrhotic rats were compared . Endotoxin decreased cardiac index in cirrhotic but not in normal rats . Although the effect of endotoxin on portal tributary blood flow was minor in all animals, a reduction in portal pressure was found in cirrhotic rats . Because the most marked hemodynamic effects were observed in cirrhotic rats, the second part of our study investigated whether platelet activating factor played a role in endotoxin-induced hemodynamic alterations in the cirrhotic model . Platelet activating factor reduced cardiac index and kidney blood flow but did not influence portal tributary blood flow . Two antagonists to platelet activating factor reduced the adverse renal blood flow lowering effects of endotoxin in cirrhotic rats . Thus, it is suggested that the hemodynamic changes observed in cirrhosis may be aggravated during acute infections . Under this condition, antagonists to platelet activating factor may be of benefit in the prevention of hemodynamic complications induced by endotoxin.

Curr Genet, 1992 Jul, 22(1), 53 - 9
Recovery of recombinant plasmids from Pleurotus ostreatus transformants; Peng M et al.; A transformation system employing selectable resistance to hygromycin B has been developed for the mushroom-forming fungus, Pleurotus ostreatus . Vector pAN7-1, a commonly used non-replicative vector for integrative transformation in fungi, yielded 5-46 resistant colonies per micrograms of DNA per 10(7) viable protoplasts . Southern blot analysis of certain transformants revealed unexpected replicative plasmids containing pAN7-1 sequences, but modified for size, methylation and restriction enzyme pattern when compared to the initial transforming vector . Two such replicative derivatives of pAN7-1 have been rescued from P . ostreatus by cloning into Escherichia coli . Rescued plasmids have been used to probe DNA from untransformed P . ostreatus in an effort to identify fungal sequences that recombined in vivo with pAN7-1 to form replicative plasmids . Such replicative sequences have been localized in high molecular weight (chromosomal) DNA of wild-type P . ostreatus . Transformation has been obtained for P . ostreatus using a rescued plasmid, thereby confirming the role of this recombinant plasmid as a shuttle vector.

J Immunol, 1992 Jul 1, 149(1), 120 - 6
Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate; Cumber AJ et al.; A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the VH domain was expressed in Escherichia coli from a modified expression plasmid containing the structural genes for the VH and VL domains derived from the anti-lysozyme hybridoma D1.3 . Chemical cross-linking between the introduced sulfhydryl groups of two FvCys fragments by means of bis-maleimidohexane was used to generate a bisFvCys conjugate . The stability of the bisFvCys conjugate and an FvCys analogue that had been reacted with N-ethyl-maleimide to block the free sulfhydryl group, FvCys(BL), were compared after 125I-labeling . The bisFvCys conjugate was completely stable to incubation in solution at 37 degrees C for 24 h whereas only 60% of the FvCys(BL) fragment remained soluble . After i.v . administration to normal Wistar rats, both Fv proteins were rapidly cleared from the circulation with biphasic kinetics that were best fitted to a two-compartment open pharmacokinetic model . The alpha-phase half-life of the bisFvCys conjugate, 0.32 h, was significantly longer than that of the FvCys(BL) fragment, 0.15 h (p less than 0.001) whereas there was no significant difference between the beta-phase half-lives, 1.4 to 1.6h . No chain cleavage or covalent attachment to serum protein was detected by SDS-PAGE analysis of serum samples . However, gel permeation HPLC revealed that both Fv proteins associated with serum proteins in vivo and in vitro.

Arch Biochem Biophys, 1992 Jul, 296(1), 137 - 43
Binding of human tumor necrosis factor alpha to multimeric complementary peptides; Fassina G et al.; A peptide with binding properties for tumor necrosis factor (TNF alpha) sequence 144-157 has been designed, using a computer-assisted method able to create peptide sequences hydropathically complementary to a given sequence . The complementary peptide was synthesized in a multimeric form starting from an octadentate polylysine core, to facilitate its immobilization and to provide interaction multivalency . Once immobilized on a solid support to prepare an affinity column, it recognized the target TNF144-157 peptide selectively from crude peptide mixtures containing TNF fragments encompassing the entire TNF alpha sequence . Similar selectivity and specificity were shown for full-length recombinant TNF alpha, allowing its purification from crude Escherichia coli extracts . The octameric complementary peptide preserved its recognition properties for TNF alpha and biotinylated TNF alpha even after coating on microtiter plates . Competitive binding occurred with unlabeled TNF alpha in the range between 0.01 and 10 micrograms/ml, in the presence of detergent such as 0.05% Tween 20 and in the presence of 1% normal goat serum . The effect of complementary peptide multimerization was evidenced by its enhanced binding affinity for TNF alpha, which exists in solution as a trimer, while the target TNF{144-157} peptide was recognized with much lower strength . The dissociation constant for interaction with TNF alpha was close to 10 nM, allowing its easy detection by solid phase assays in concentrations as low as 10 pmol/ml.

Virology, 1992 Jul, 189(1), 150 - 60
Pathways of viral gene expression during acute neuronal infection with HSV-1; Margolis TP et al.; Pathways of viral gene expression were investigated during the acute phase of sensory ganglionic infection with HSV-1 . To facilitate these studies we constructed KOS/62-3, an HSV-1 vector in which the Escherichia coli lac-Z gene was inserted behind both copies of the promoter for the viral latency-associated transcripts . Following footpad inoculation of mice with the virus, acutely infected dorsal root ganglion (DRG) neurons were assayed by dual immunofluorescence for the presence of beta-galactosidase and HSV viral antigens . Most infected neurons stained for either beta-galactosidase or viral antigens . Less than 0.2% of neurons staining for viral antigens also expressed beta-galactosidase, and less than 10% of neurons expressing beta-galactosidase also stained for viral antigen . As a consequence of these findings, we propose that there are essentially two populations of HSV-infected neurons during the acute phase of ganglionic infection . In one population of neurons there is abundant viral protein synthesis but minimal transcription of latency-associated transcripts, whereas in a second population of neurons viral gene expression is severely restricted except for the synthesis of latency-associated transcripts . Since DRG neurons are a heterogeneous population of cells, we further sought to determine whether either pathway of gene expression was more likely to occur in a particular neuronal phenotype . To accomplish this, antibodies were used to characterize the DRG neuronal phenotypes acutely infected with the virus . The results indicated that the pathway of neuronal infection characterized by transcription of abundant latency-associated transcripts and minimal viral protein synthesis was much more likely to occur in DRG neurons expressing the cellular antigen SSEA-3 . These data indicate that the neuron plays a major role in regulating the outcome of infection with HSV . Finally, we sought to determine whether DNA replication occurs in the course of establishment of a latent infection . We found that the DNA content of neurons latently infected with KOS(M) strain HSV was not affected by treatment with nucleotide analogues during the acute phase of ganglionic infection, suggesting that viral DNA replication does not occur during the establishment of latent infection.

Proteins, 1992 Jul, 13(3), 206 - 22
A multiple-start Monte Carlo docking method; Hart TN et al.; We present a method to search for possible binding modes of molecular fragments at a specific site of a potential drug target of known structure . Our method is based on a Monte Carlo (MC) algorithm applied to the translational and rotational degrees of freedom of the probe fragment . Starting from a randomly generated initial configuration, favorable binding modes are generated using a two-step process . An MC run is first performed in which the energy in the Metropolis algorithm is substituted by a score function that measures the average distance of the probe to the target surface . This has the effect of making buried probes move toward the target surface and also allows enhanced sampling of deep pockets . In a second MC run, a pairwise atom potential function is used, and the temperature parameter is slowly lowered during the run (Simulated Annealing) . We repeat this procedure starting from a large number of different randomly generated initial configurations in order to find all energetically favorable docking modes in a specified region around the target . We test this method using two inhibitor-receptor systems: Streptomyces griseus proteinase B in complex with the third domain of the ovomucoid inhibitor from turkey, and dihydrofolate reductase from E . coli in complex with methotrexate . The method could consistently reproduce the complex found in the crystal structure searching from random initial positions in cubes ranging from 25 A to 50 A about the binding site . In the case of SGPB, we were also successful in docking to the native structure . In addition, we were successful in docking small probes in a search that included the entire protein surface.

J Virol, 1992 Jul, 66(7), 4413 - 26
Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins; Rempel RE et al.; The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases . In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication . In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication . We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented . Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures . These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors . To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli . The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro . The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1 . In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C . No null mutants were isolated . These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C.

J Virol, 1992 Jul, 66(7), 4117 - 25
Recombinant human adenoviruses containing hybrid adenovirus type 5 (Ad5)/Ad12 E1A genes: characterization of hybrid E1A proteins and analysis of transforming activity and host range; Jelinek T et al.; Hybrid adenovirus type 12 (Ad12)/Ad5 E1A genes were constructed by homologous recombination in Escherichia coli, a technique which offers several advantages over conventional mutagenesis for genetic analysis of proteins . In particular, functional differences between the proteins can be mapped by correlating the replacement of specific sequences with the acquisition of new properties, and there is no requirement for common unique restriction sites or polymerase chain reaction strategies to construct the hybrids . Recombinant adenoviruses expressing these hybrid E1A proteins were capable of replicating efficiently in HeLa cells, with the exception of one construct which contained a hybrid transactivation domain . The transforming activity of the hybrid E1A constructs was assayed by DNA transfection of primary baby rat kidney cells . Plasmids containing Ad12 E1 were approximately 20-fold less efficient at transformation than those with E1 of Ad5, and it was found that two regions in exon 1 of E1A mediate this difference . No differences were found in the abilities of any hybrid E1A proteins to bind to cellular proteins previously determined to be important for transformation by E1A.

DNA Cell Biol, 1992 Jul-Aug, 11(6), 489 - 96
Expression of human 60-kD heat shock protein (HSP60 or P1) in Escherichia coli and the development and characterization of corresponding monoclonal antibodies; Singh B et al.; Human P1 protein, which is the homolog of the 60- to 65-kD heat shock "common" antigenic protein of numerous pathogenic organisms (synonyms: HSP60, GroEL homolog, or chaperonin), has been expressed to high level in Escherichia coli cells . A large number of well-characterized deletions of this protein spanning the entire sequence have been constructed and expressed . Methods to purify recombinant human HSP60 protein and its deletions from E . coli have been worked out . In addition, monoclonal antibodies to the human HSP60 protein have been raised and partially characterized . The availability of these materials should greatly aid in understanding the role of this highly conserved and immunologically important protein in autoimmune diseases and in cell structure and function.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 175 - 80
Sequence and characterization of the gcpE gene of Escherichia coli; Baker J et al.; In Escherichia coli, the gene gcpE encodes a protein of unknown function and lies immediately upstream of the hisS gene for histidyl-tRNA synthetase . The nucleic acid sequence of gcpE predicts an open reading frame for a protein of 40,681 Da . The probable transcription terminator of gcpE overlaps the hisS promoter . Haploid cells containing a disrupted gcpE could not be obtained, suggesting that the gcpE gene is essential.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 15 - 8
Transcription of glpT of Escherichia coli K12 is regulated by anaerobiosis and fnr; Wong KK et al.; The transcriptional expression of the sn-glycerol 3-phosphate transport gene, glpT, was studied with a glpT-lac fusion contained on a low-copy-number plasmid pFZY1 . The fusion was isolated during a 'shot gun' cloning of promoters expressed under anaerobic growth conditions . It was shown that glpT was induced six-fold by anaerobiosis . Furthermore, aerobic expression of glpT was induced by the substrate glycerol 3-phosphate, while the anaerobic expression of glpT was decreased by nitrate, glucose and a fnr mutation . However, nitrate repression on glpT expression was relieved if the upstream region from -290 of the transcription site was removed.

Zentralbl Bakteriol, 1992 Jul, 277(2), 170 - 8
A sensitive method for the detection of enterotoxigenic Escherichia coli by the polymerase chain reaction using multiple primer pairs; Abe A et al.; In this study, a polymerase chain reaction (PCR) method has been developed for the detection of enterotoxigenic Escherichia coli (ETEC) . Three different sets of oligonucleotide primers synthesized were used to amplify the enterotoxin genes of heat-labile (LTh) and heat-stable (STIa and STIb) enterotoxins of ETEC . These primers amplified a 627, 240, or 169 base pair (bp) DNA fragment from LTh, STIa and STIb gene, respectively, of the reference ETEC strains . The addition of RNase A (10 micrograms/ml) to the PCR reaction solution diminished nonspecific amplification of DNA fragments other than the enterotoxin genes . Five types of ETEC strains corresponding to the LTh, STIa, STIb, LTh-STIa, or LTh-STIb genotypes were distinguished by a single procedure of PCR using the mixture of the three sets of primers . PCR, hybridization, and conventional methods were subjected to one hundred stool specimens from diarrheal patients . It was found that PCR was the most sensitive method among them . These results suggested that PCR with triple primer pairs would be useful for the laboratory diagnosis of ETEC in the stool specimens.

AIDS Res Hum Retroviruses, 1992 Jul, 8(7), 1301 - 10
Mapping of immunodominant epitopes of the HIV-1 and HIV-2 integrase proteins by recombinant proteins and synthetic peptides; Vinga-Martins C et al.; Different parts of the human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2) integrase proteins were expressed as TrpE fusion proteins in Escherichia coli and used to screen human sera . In the immunoblot, all HIV/integrase-positive human sera tested reacted with the carboxy-terminal third of the integrase protein . Furthermore, they crossreacted with the same part of the heterologous protein . Half (50%) of the HIV-1/integrase-positive sera additionally detected antigenic epitopes in the amino-terminal third of the HIV-1 protein . Two of the recombinant proteins were used to generate polyclonal rabbit sera, which react with type-common epitopes of both integrase proteins . To map the B-cell epitopes of the HIV integrase proteins in more detail, overlapping decapeptides representing the entire integrase proteins of HIV-1 and HIV-2 were synthesized and used in a pin-based oligopeptide ELISA to scan human sera . This method can define three potential immunogenic epitopes of the HIV-1 integrase and one potential epitope of the HIV-2 integrase . The immunodominant epitopes of the HIV-1 integrase, one localized in the amino-terminal (IDKAQDEHEKYHSNWRAM), one in the central (QMAVFIHNFKRKGGIGGY), and one in the carboxy-terminal (AVVIQDNSDIKVVPRRK) part of the protein were synthesized as oligopeptides and used to test a larger panel of human sera in ELISA (156 HIV-1+ sera and 104 HIV-1- sera) . The amino- and the carboxy-terminal epitopes were of equivalent reactivity, while the central part of the HIV-1 integrase seems to be less immunogenic . Nearly 90% of the HIV-1/integrase-positive human sera could be detected by a combination of these three peptides.

Protein Eng, 1992 Jul, 5(5), 461 - 6
Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter; Shuttleworth WA et al.; 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of the Escherichia coli aroA gene, has been overproduced in E . coli BL21(lambda DE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of approximately 50% of total cell protein . EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as Roundup . A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE) . The integrity of the protein has been compared with previously characterized protein from E . coli AB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and 13C-NMR spectroscopy . This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.

Protein Eng, 1992 Jul, 5(5), 455 - 9
Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli; Taylor MA et al.; For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli . This inactive precursor can subsequently be processed to yield active enzyme . Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases . A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E . coli using a T7 polymerase expression system . Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6 . A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6 . Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6 . Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine . Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA . This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.

Protein Eng, 1992 Jul, 5(5), 447 - 53
Complex additivity of the effects of suppressor mutations in differing protein environments; Dunn IS et al.; In the alpha-complementation of beta-galactosidase, a defective beta-galactosidase protein interacts with an autologous peptide fragment (alpha-peptide) to restore enzymatic activity . Within a specific site of a defective alpha-peptide we have previously isolated a large number of mutations, many of which suppress the functional defect . The alpha-peptide was originally defective due to both insertional and substitutional sequence alterations near its N-terminus, which provided an increase in the sensitivity of detection of (suppressor) secondary mutations which conferred improved function . We have now studied the effects of the suppressor mutations when the primary deleterious mutations are sequentially reversed . This was done in intact beta-galactosidase, as we have shown that mutations in the alpha-peptide have related functional effects in the whole protein . Evidence was obtained showing that the effects of at least some suppressor mutations were not simply additive when the mutations are placed into the original wild-type protein environment . One suppressor appeared to function less effectively in the normal environment, while another when tested in the same manner functioned at a relatively increased level . This failure to show simple additivity may be attributable to the physical proximity of the original defective mutations and the introduced suppressors . Nevertheless, even in such cases it may be feasible to use a defective protein as a sensitive starting point for the identification of mutations which improve the wild-type protein.

Protein Eng, 1992 Jul, 5(5), 441 - 6
Mutant forms of beta-galactosidase with an altered requirement for magnesium ions; Dunn IS et al.; By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants . The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system . We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator . In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+ . However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions . In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase . We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases . Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.

Protein Eng, 1992 Jul, 5(5), 427 - 31
Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini; Horlick RA et al.; A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described . In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem . Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein . By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively . The name 'permutein' is proposed to describe proteins generated by this technology . The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta . The approach should be useful to define further the structural features of this protein that are important for its function.

Anal Biochem, 1992 Jul, 204(1), 204 - 9
Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect; Waters TR et al.; A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed . The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate . The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases . Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis . Both of the hexameric products are single stranded under the assay conditions . The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect . This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.

Anal Biochem, 1992 Jul, 204(1), 190 - 7
Analysis of human synovial fluid phospholipase A2 on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric assay suitable for a microtiterplate reader; Reynolds LJ et al.; The development of a reliable assay for human synovial fluid phospholipase A2 (HSF PLA2) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors . This enzyme behaves differently from other extracellular PLA2s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate . We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors . The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 mumol min-1 mg-1 . The substrate concentration curve fits a Hill equation with an apparent Km of 500 microM and a Hill coefficient of two . The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity . The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity . Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC50 of 18 microM.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1413 - 20
Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli; Xue GP et al.; A cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli . Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose . Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC . Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose . Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed beta-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose . Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme . Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases . Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N . patriciarum was induced by cellulose.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1399 - 408
Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis; Milano A et al.; The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms . The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined . Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively . The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity . A small subunit has not been identified for either of the S . platensis AHS isoenzymes . Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1379 - 86
Isolation and characterization of a conjugative plasmid from Legionella pneumophila; Mintz CS et al.; The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied . To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients . Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe . The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L . pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli . Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L . pneumophila plasmids . There was no detectable DNA homology between pCH1 and L . pneumophila chromosomal DNA . Additional mating experiments revealed that pCH1 was unable to mobilize the L . pneumophila chromosome . The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis . These results suggest that pCH1 does not contribute to the ability of L . pneumophila to enter or grow within eukaryotic cells.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1345 - 52
A survey of Col plasmids in natural isolates of Escherichia coli and an investigation into the stability of Col-plasmid lineages; Riley MA et al.; A survey of colicins in the ECOR reference collection of Escherichia coli is presented . Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E . coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts . Multiple representatives of two Col plasmids, low-molecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis . These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1301 - 7
Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath); West CA et al.; Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol . sMMO is comprised of three components; A, B and C . Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow . The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli . A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system . Upon induction, E . coli expressed protein B which was fully functional after purification . The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon) . Protein C expressed in E . coli was also found to be functional . This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.

Prostaglandins Leukot Essent Fatty Acids, 1992 Jul, 46(3), 219 - 22
Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli; Ensor CM et al.; NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins . The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein . The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence . The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter . Extracts from E . coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG) . Crude extracts from E . coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum . The specific activity in E . coli extracts was several hundred-fold higher than that seen in extracts from human placenta.

Mol Endocrinol, 1992 Jul, 6(7), 1142 - 52
Thyroid hormone alters in vitro DNA binding of monomers and dimers of thyroid hormone receptors; Ribeiro RC et al.; T3 binds to intranuclear thyroid hormone receptors (TRs) on target DNA elements and exerts profound influences on gene expression by mechanisms not yet characterized . We used gel shift assays and cross-linking experiments to demonstrate that T3 greatly induced the monomeric binding of the hTR beta produced in Escherichia coli to DNA . T3 also increased the gel mobility of these monomer-DNA complexes suggesting they undergo a ligand-induced conformational change . This effect did not depend on the orientation and spacing of the half-site motifs within the DNA structure . In contrast, T3 had diverse effects on the dimeric interaction . T3 increased the dimeric interaction to the palindrome GGTCA.TGACC (an effect lost by spacing the half-sites with 3 base pairs) and decreased the dimeric interaction to the inverted palindrome containing the TGACC.GGTCA motif . Scatchard analyses indicated that the T3 enhancement on binding was due to an increase in the number of TR with high affinity DNA-binding activity and not by increasing the affinity of TR that could bind to DNA . The effects of various T3 analogs were directly related to their affinities for the TR . These ligand effects on in vitro TR-DNA binding may reflect mechanisms by which T3 regulates transcription in vivo.

Mol Microbiol, 1992 Jul, 6(14), 1969 - 79
The divergent promoters mediating transcription of the par locus of plasmid RP4 are subject to autoregulation; Eberl L et al.; The partitioning region of broad-host-range plasmid RP4 contains four genes (parA, parB, parC, and parD) that encode products essential for partition activity . Two divergently arranged promoters located in the intercistronic region between parC and parD mediate transcription of these genes . The transcriptional initiation sites for both promoters were determined by primer extension . Transcriptional fusions were used to show that parA, parB, and parC are combined in an operon, while parD constitutes a separate transcription unit . Both parCBA (genes in order of transcription) and parD are negatively autoregulated at the level of transcription by the gene products of parA and parD, respectively . parD promoter mutants which have become insensitive to repression by parD were isolated . Comparison of wild type and the mutant parD promoter sequences indicated that three short repeats are likely involved in the negative regulation of this promoter . Potentially these sequence elements comprise target sites for the ParD protein.

J Periodontol, 1992 Jul, 63(7), 626 - 32
The effects of the Nd:YAG laser on in vitro fibroblast attachment to endotoxin-treated root surfaces; Trylovich DJ et al.; The purpose of this study was to evaluate the effects of the Nd:YAG laser on in vitro fibroblast attachment to endotoxin-treated root surfaces and to describe any laser-induced cementum surface alterations . Thirty 4 mm x 4 mm cementum segments were obtained from unerupted third molars . The treatment groups were as follows: 1) control, healthy root segment; 2) non-lased, endotoxin treated; and 3) lased, endotoxin treated . The endotoxin treated roots were soaked in E . coli 055:B5 lipopolysaccharide (556 EU/ml) for 72 hours . The lased, endotoxin-treated root segments were treated with a Nd:YAG laser using a 320 microns contact optic fiber handpiece with an energy setting of 80 mJ at 10 pulses per second for one minute . The root segments were subsequently placed in fibroblast culture dishes for 40 hours and then prepared for scanning electron microscopy (SEM) observation . SEM examination revealed two different types of attachment: flat and round . Flat cells represented firmly attached cells due to well-defined points of attachment and numerous lamellapodia . Round cells possessed few attachment processes and were, therefore, considered poorly attached . The lased, endotoxin-treated root segments had significantly decreased numbers of flat fibroblasts versus the control and non-lased, endotoxin-treated root segments . The absence of flat fibroblasts in the laser treated root segments was a consistent finding . The non-lased, endotoxin-treated root segments had significantly increased numbers of round fibroblasts versus the control and lased, endotoxin treated groups . The lased root segments exhibited surface alterations which included charring, crater formation, cementum meltdown, and tracking.(ABSTRACT TRUNCATED AT 250 WORDS)

J Appl Physiol, 1992 Jul, 73(1), 324 - 8
Reversal of hyperdynamic response to continuous endotoxin administration by inhibition of NO synthesis; Meyer J et al.; Septic shock is characterized by an increase in cardiac output and a fall in systemic vascular resistance index and mean arterial pressure . Endotoxin alters the smooth muscle function of blood vessels, probably by means of an increased production of the potent vasodilator nitric oxide (NO) . The present study was accomplished to determine how the inhibition of NO synthesis influences cardiovascular performance in an ovine model of hyperdynamic endotoxemia . Endotoxemia was induced in five range ewes (41 +/- 2 kg) by continuous infusion of Escherichia coli endotoxin (LPS, 10 ng.kg-1.min-1) over the entire study period . After 24 h of LPS infusion, cardiac output increased from 5.2 +/- 0.3 to 7.9 +/- 0.6 (SE) 1/min (P less than 0.05) and mean arterial pressure and systemic vascular resistance index fell from 92 +/- 5 to 79 +/- 6 mmHg (P = 0.08) and from 1,473 +/- 173 to 824 +/- 108 dyn.s.cm-5.m2 (P less than 0.05), respectively . The pulmonary shunt fraction increased from 0.23 +/- 0.03 to 0.32 +/- 0.03 (P less than 0.05) . The intravenous administration of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (25 mg/kg) 24 h after the start of the LPS infusion changed these values to approximately baseline levels over the subsequent 4 h . Although N omega-nitro-L-arginine methyl ester increased pulmonary arterial pressure and pulmonary vascular resistance (P less than 0.05), right and left ventricular stroke volume index showed no significant changes . It is concluded that NO has a major function in cardiovascular performance in endotoxemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechniques, 1992 Jul, 13(1), 56 - 8, 60, 62
DNA cassettes containing the origin of transfer (oriT) of two broad-host-range transfer systems; Hengen PN et al.; Plasmid constructs are described that carry retrievable DNA cassettes containing the origin of transfer region (oriT) from two broad-host-range plasmids . Restriction of these high copy number plasmids with any one of a variety of enzymes yields a linear DNA fragment of convenient size containing the oriT region of either pCUI or RK2 . This DNA can be ligated into any vector or recombinant plasmid containing a compatible enzyme site and can be easily identified by size on an agarose gel . Any plasmid can therefore be mobilized using a number of helper strains or conjugative plasmids derived from the parental plasmids . In addition, the cassettes can be used for a variety of genetic manipulations including "selectable" linker mutagenesis.

Biochem Int, 1992 Jul, 27(2), 221 - 9
Novel fractionation of proteins using novobiocin as a precipitating agent: application to ribosomal proteins from the archaebacterium Sulfolobus acidocaldarius and Escherichia coli; Srinivas R et al.; A fractionation method involving precipitation of proteins with novobiocin has been developed to separate ribosomal proteins of the archaebacterium Sulfolobus acidocaldarius . Four groups of proteins were obtained corresponding to those precipitated with 0.2, 0.5 and 1.0 mg/ml novobiocin and the proteins soluble at 1.0 mg/ml novobiocin . The same procedure was also successful for the fractionation of ribosomal proteins from Escherichia coli . Two dimensional gel electrophoresis showed that proteins present in each group were distinct with only a minor contamination of proteins from other groups . Our results indicate that proteins with similar charge and molecular weight can be separated by this method . This procedure may have applications for the fractionation of other complex protein mixtures.

Mol Biochem Parasitol, 1992 Jul, 53(1-2), 223 - 31
The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei; Fairlamb AH et al.; D,L-dihydrolipoamide and D,L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10(9) and 4.51 x 10(9) M-1, respectively . These complexes possess 6-membered cyclic dithioarsenite rings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500-fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione . L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate . The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 +/- 4.3 and 9.7 +/- 2.9 pmol (10(8) cells)-1, respectively) . The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 +/- 1.4 and 47.8 +/- 3.7 mU mg-1, respectively) . However, the Km for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains . Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane . These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs.

Mol Biochem Parasitol, 1992 Jul, 53(1-2), 159 - 67
Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris; Eschenbacher KH et al.; Using polyadenylated RNA isolated from Sarcocystis muris cyst merozoites, we have constructed a cDNA library in the expression vector lambda ZAP . Immunoscreening with monoclonal and polyclonal antibodies directed against a 31-kDa surface antigen of S . muris {1} yielded a number of clones with insert sizes ranging between 1.1 kb and 1.3 kb . An additional clone with an insert length of 1.55 kb was isolated by screening with a labeled DNA probe derived from one of the cDNA clones . The cDNA sequence was found to contain an open reading frame specifying a polypeptide of 280 amino acids with a predicted size of 29.7 kDa . The deduced amino acid sequence is rich in serine and threonine (22%) and harbors a hypothetical N-terminal signal peptide sequence as well as a C-terminal glycosyl phosphatidylinositol anchor attachment site . The predicted amino acid sequence has been confirmed by peptide sequencing and an analysis of the overall amino acid composition of the 31-kDa protein . A recombinant protein was obtained which was recognized by the polyclonal antibodies directed against the 31-kDa antigen . Antiserum raised against the purified fusion protein specifically reacted with a 31-kDa protein from S . muris cystozoites . Southern blot analysis indicated that the corresponding gene exists as a single copy within the S . muris genome.

J Dairy Sci, 1992 Jul, 75(7), 1826 - 34
Kinetics and characteristics of bovine neutrophil alkaline phosphatase during acute Escherichia coli mastitis; Heyneman R et al.; Alkaline phosphatase activity of isolated bovine blood neutrophils was investigated before and during experimentally induced Escherichia coli mastitis . Activities markedly increased 1 wk after infection in neutrophils of cows suffering from moderate or from severe disease . Elevated neutrophil alkaline phosphatase activity did not correlate with a specific stage of maturating postmitotic neutrophils appearing in circulation during mastitis . Neutrophil alkaline phosphatase from healthy cows before infection and from mastitic cows was characterized by means of thermostability, specific inhibitor patterns, slab gel electrophoresis, and kinetic parameter analysis . The leukocyte enzyme from healthy and mastitic cows displayed very similar characteristics, suggesting that the increase in activity during mastitis is most probably related to the enhanced expression of the normal alkaline phosphatase enzyme.

Am J Vet Res, 1992 Jul, 53(7), 1145 - 8
Myoelectric activity of the small intestine in enterotoxin-induced diarrhea of calves; Roussel AJ et al.; Electrodes were surgically implanted at 15-cm intervals in the jejunum and ileum of 4 healthy neonatal calves so that myoelectric activity could be recorded on 2 consecutive days . On the first day, each calf received a control treatment, and myoelectric activity was recorded for 340 minutes . Phase I was recorded for a mean of 175.8 +/- 22.8 minutes (51.5%), phase II for 124 +/- 27.4 minutes (36.5%), and phase III for 40.3 +/- 6 minutes (11.9%) . On the second day, each calf was treated with approximately 200 micrograms of heat-stable enterotoxin (STa) of Escherichia coli orally . All calves developed diarrhea after the administration of STa . Phase I was recorded for a mean of 92.5 +/- 42.3 minutes (27.2%), phase II for 227.3 +/- 52.5 minutes (66.9%), and phase III for 20.3 +/- 11.4 minutes (6.0%) . Increase in phase II and decrease in phases I and III after STa administration were significant (P less than 0.05) . Duration of the migrating myoelectric complex was longer after STa administration (median, 64 minutes), compared with the control treatment (median, 54 minutes) . Minute rhythms, recorded on the day of toxin administration, ranged from 49 to 153 minutes . There was no difference between the number of migrating action potential complexes on the control days (range, 1 to 10), compared with those on treatment days (range, 1 to 14) . These findings are suggestive that enterotoxin-induced diarrhea of calves is accompanied by increased total spiking activity and minute rhythms in the distal portion of the jejunum and ileum.

Mol Gen Genet, 1992 Jul, 234(1), 43 - 8
A molecular marker to select for freezing tolerance in Gramineae; Houde M et al.; We isolated, and expressed in Escherichia coli, a gene (Wcs120) that is strongly induced during cold acclimation of wheat . The gene product was purified and used to produce antibodies . Immunoblotting experiments with the anti-WCS120 antibody identified several cold-induced proteins named FTMs for Freezing Tolerance Markers since they are associated with the development of freezing tolerance . This protein family was found to be coordinately regulated specifically by low temperature, highly hydrophilic, stable to boiling, and to have a pI above 6.5 . The accumulation kinetics during the acclimation period indicated a positive correlation with the capacity of each genotype to develop freezing tolerance . Accumulation of the proteins was higher in the freezing-tolerant genotype than in the less tolerant one . In addition, their accumulation was more pronounced in the crown and leaf tissues compared with roots, confirming a relationship to the capacity of the different tissues to develop freezing tolerance . Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals . The antibody did not cross-react with any of the non-cereal species examined . The anti-FTMs antibody represents a potential tool for breeders to select for freezing tolerance traits in the Gramineae.

Mol Gen Genet, 1992 Jul, 234(1), 155 - 63
A topological model for the haemolysin translocator protein HlyD; Schulein R et al.; A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD . Active HlyD-LacZ fusion proteins were only generated when lacZ was fused to hlyD within the first 180 bp (60 amino acids) . HlyD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD) . Active insertions of phoA into the middle region of hlyD were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques . A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein . In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound . Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins . These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1992 Jul, 234(1), 14 - 21
Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli; Hansen FG et al.; Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations . The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene . The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene . Several of the mutant dnaA genes were found to have two mutations . The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution . The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA211 each had only one . The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations . Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains . Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein.

J Biol Regul Homeost Agents, 1992 Jul-Sep, 6(3), 99 - 102
Rapid high level production and purification of recombinant murine and human interferons alpha from Escherichia coli; Boyer SJ et al.; The availability of large quantities of pure interferon alpha (IFN-alpha) subtypes for in vivo studies has often proved difficult . This paper presents details on the use of the commercially available pGEX expression system for the production and purification of milligram (mg) quantities of recombinant Murine (Mu) and Human (Hu) IFNs-alpha-1 in Escherichia coli . Initially a fusion product is made which can be rapidly purified on a glutathione-sepharose 4B affinity matrix . Biologically active IFN-alpha can then be released from the matrix by cleavage with the restriction protease activated factor X (FXa+7,++) . Routine yields of the final products were in the range of 0.5 to 2.0 mg/l of original culture.

Reg Immunol, 1992 Jul-Aug, 4(4), 255 - 61
Diminished immunoglobulin synthesis after stimulation of mononuclear cells from periodontal disease tissue; Lundqvist CA et al.; Gingival mononuclear cells from patients with adult periodontitis were cultured to determine the potential for IgG production . All samples (N = 27) showed IgG synthesis . Some samples demonstrated IgG synthetic activity over the entire period in culture, often with maximum synthesis after 8 days . Other samples showed IgG synthesis during the first half of the culture period and then little detectable production for the remainder . Cells were either untreated or treated with one of several different known mitogens during the culture period . Total IgG synthesis by peripheral blood lymphocytes was enhanced in the presence of pokeweed mitogen, E . coli lipopolysaccharide and killed . A . actinomycetemcomitans . In contrast, IgG synthesis by gingival cells in the presence of these same additives was significantly reduced when compared to gingival cell synthesis in the absence of mitogens; suggesting the presence of suppression in this system . These differences in responsiveness may be attributable to the unique combination of T cells found in the gingival tissues of patients with periodontal diseases . The patterns of IgG synthesis by gingival cells were different from those of peripheral blood cells from the same patient . This finding verified the distinctiveness of local immunoregulatory mechanisms in periodontal disease tissue from those found systemically.

Res Microbiol, 1992 Jul-Aug, 143(6), 605 - 13
Genetic approaches to cell biology and metabolism of spirochetes; Penn CW et al.; Genetic analysis and methodology have only comparatively recently been applied to the study of spirochetes . Although genetic transfer procedures for spirochetes are not widely available, there are several examples of progress in genetic analysis of spirochetes by other approaches . Some examples of these approaches are the following . 1) Genes for synthetic pathways in Treponema and Leptospira have been cloned by complementation of Escherichia coli serving as plasmid hosts . 2) The OspA protein of Borrelia burgdorferi has been overexpressed in E . coli without the signal peptide; the recombinant product has been suitable for circular dichroism as well as other biochemical analyses . 3) The heat shock proteins of B . burgdorferi are homologous to heat shock proteins of E . coli . 4) Enzyme activity profiles of B . burgdorferi and other spirochetes show strain heterogeneity and also indicate which biosynthetic and enzymatic activities are conserved within different spirochetes . 5) The gene organization of rRNA genes have revealed differences between spirochetes and other types of bacteria.

Res Microbiol, 1992 Jul-Aug, 143(6), 583 - 96
Antigenic variation and strain heterogeneity in Borrelia spp; Wilske B et al.; Antigenic variation and strain heterogeneity have been demonstrated for the pathogenic Borrelia species, i.e . B . burgdorferi and the relapsing fever borreliae . In relapsing fever, new borrelia serotypes emerge at a high rate spontaneously, a mechanism that is caused by DNA rearrangements on linear plasmid translocating genes coding for variable major proteins from previous silent to expression sites (i.e . from inner sites to telomeric sites of the plasmid) . As a result of this variation, the borreliae escape the immune response of the host, thus leading to the relapse phenomenon . In B . burgdorferi, which is the causative agent of the multisystem disorder Lyme borreliosis, there is also a growing body of findings that antigenic variation is involved in pathogenesis of the disease . Phenotypic variation of strains in vitro concerns the size and the amount of surface-associated proteins (OspA, OspB and pC) . There are indications that OspA and OspB truncations are due to deletions within the ospAB operon caused by recombination events, and that OspA/OspB-less mutants lack the 49-kb plasmid that bears the ospAB operon . With the increasing number of isolates obtained from various geographic and biological sources, it became apparent that B . burgdorferi is immunologically and genetically more heterogeneous, as previously believed . The major outer surface proteins OspA and OspB (which have been efficient antigens in vaccine studies) are heterogeneous at a genetic level . The same degree of genetic non-identity was observed for the pC protein . Other proteins like flagellin and the highly specific immunodominant p100 range protein show a lower degree of non-identity . Recombinant OspA, pC, p100 range protein and flagellin have been hyperexpressed in E . coli and these proteins are immunologically reactive . This allows further research for development of vaccines and diagnostic tools . B . burgdorferi isolates have been investigated with genotyping (DNA hybridization, PCR and 16S rRNA analysis) as well as serotyping by various authors . Comparison of the different methods has shown good agreement when the same strains have been investigated . No correlation could be found between different phenotypic and genotypic groups with respect to the ability to cause arthritis in SCID mice . A serotyping system based on immunological differences in OspA detected by a panel of monoclonal antibodies has been proposed . Serotyping a large number of B . burgdorferi isolates has shown a striking predominance of the OspA serotype 2 among European isolates from human skin, in contrast to isolates from ticks or CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Mikrobiologiia, 1992 Jul-Aug, 61(4), 666 - 71
{Features of the initial stage of surface colonization by Eshcherichia coli cells as a function of their mobility and chemotaxis}; Voskun SE et al.; Dependence of adhesion and colonization of hydrophilic and hydrophobic surfaces by Escherichia coli strains with different mobility and chemotaxis was studied using E . coli mot+che+, E . coli mot+che-, E . coli mot-che+ . Primary adhesion was shown to correlate with mobility of cells and hydrophilic/hydrophobic character of their surface . Secondary adhesion correlated in addition with chemotactic characteristics of bacteria . E . coli populations were shown to vary in electrophoretic mobility and cells capability for adhesion and chemotaxis.

Mikrobiologiia, 1992 Jul-Aug, 61(4), 598 - 603
{Dynamics of the population structure of the Escherichia coli recombinant strain during continuous culture}; Popova LIu et al.; The populational structure of the Escherichia coli strain Z905 containing the recombinant plasmid with the phenotype AprLux+ was studied in chemostat . It was shown that the stability of the ratio of plasmid containing cells and cells without plasmids depends in the first place on the presence of the selective factor (ampicillin) in the medium and on the sources of carbon and energy limiting growth.

Biull Eksp Biol Med, 1992 Jul, 114(8), 199 - 201
{The systems of the genetic regulation of plasmid transfer fin K, fin L, fin M and fin N}; Maksimenko LV et al.; F-like plasmids pAP19-1::Tn9, pAP20::Tn9, pAP22-1::Tn1, pAP27 characterized by the presence of unique genetic plasmid transfer regulatory systems in their genomes have been found . These systems were named fin K, fin L, fin M, finN, consequently . They were characterized from the point of view of specificity of their action on F-factor and F-like conjugative function . Dependence of fin N-system expression on host-cell and on the order of plasmid entering into host-cell was shown.

Biull Eksp Biol Med, 1992 Jul, 114(8), 196 - 8
{The genome localization of the plasmid pAP27 fin N system}; Maksimenko LV et al.; Plasmid transfer genetic regulatory system fin N of F-like plasmid pAP27 have been localized on BamHI-restriction fragment f3 (length 8.7 kb) . Plasmid pUC19 Fin-activity have been cleared up and characterized from the point of view of its specificity of action on some plasmid transfer functions.

Biull Eksp Biol Med, 1992 Jul, 114(8), 179 - 81
{The production and characteristics of an interleukin-6-dependent hybridoma}; Kliushnenkova EN et al.; The interleukin-6-(IL-6)-alpha dependent B-cell heterohybridomas were obtained by the fusion of X65.Ag8.653 cells with spleen cells from August rats immunized with lipopolysaccharide E . coli . One of these hybridomas (D6C8) was found to be most dependent on IL-6 for its surviving and growth . Human recombinant IL-1 beta and tumor necrosis factor-alpha could not induce the in vitro growth of this cell line . Presence of elevated level of IL-6 was demonstrated in the sera of patients with rheumatoid arthritis . A specific and sensitive detection of the IL-6 activity in test samples makes it possible to study the presence and role of IL-6 in various immunological disorders.

Bioessays, 1992 Jul, 14(7), 445 - 54
How the EcoRI endonuclease recognizes and cleaves DNA; Heitman J; One popular recombinant DNA tool is the EcoRI endonuclease, which cleaves DNA at GAATTC sites and serves as a paradigm for sequence specific DNA-enzyme interactions . The recently revised X-ray crystal structure of an EcoRI-DNA complex reveals EcoRI employs novel DNA recognition motifs, a four alpha-helix bundle and two extended chains, which project into the major groove to contact substrate purines and pyrimidines . Interestingly, pyrimidine contacts had been predicted based on genetic and biochemical studies . Current work focuses on the EcoRI active site structure, enzyme and substrate conformational changes during catalysis, and host-restriction system interactions.

Mol Biol (Mosk), 1992 Jul-Aug, 26(4), 772 - 9
{Expression of orthopoxvirus DNA sequences in Escherichia coli cells}; Shchelkunov SN et al.; We observed the expression of recombinant plasmids genes containing ectromelia virus DNA fragments in E . coli minicells . Using plasmids with vaccinia or ectromelia viruses DNA fragments inserted upstream of lacZ gene we showed that certain orthopoxvirus genome fragments carry out a promoter-like function in bacterial cells.

Med Parazitol (Mosk), 1992 Jul-Aug, (4), 5 - 7
{The characteristics of cryptosporidiosis prevalence in Nizhni Novgorod Province}; Romanova TV et al.; A total of 4070 patients with acute intestinal diseases were tested for cryptosporidiosis in 1989, 1806 of these without diarrhea (controls), 286 of them working at slaughter houses, milk-packing plants and dairy plants, 192 subjects with a history of contacts with cryptosporidiosis patients in the focus of the disease, and 7 animals . Cryptosporidiosis was responsible for 3.2% of acute intestinal conditions in children; cryptosporidia oocysts were not detected in adults or healthy subjects . The high risk group invasion has made up 2.7% . Two cases of the invasion were detected among subjects with a history of contacts with cryptosporidiosis patients, this making up 1.04%, none of them presenting with any clinical symptoms of the invasion.

Kansenshogaku Zasshi, 1992 Jul, 66(7), 950 - 5
{Isolation of vero-cytotoxin-producing Escherichia coli from cattle and serotyping and toxin-typing of the isolated strains}; Hirata K et al.; Two hundred and sixty-six piglets with diarrhea (from 4 farms), 73 healthy pregnant pig (from 2 farms), 27 calves with diarrhea (from 9 farms) and 47 healthy milk cows (from 1 farm) were examined for Vero-cytotoxin-producing Escherichia coli (VTEC), and 52, 11, 15 and 67 strains of VTEC were isolated from 17 piglets, 11 pregnant pigs, 6 calves and 23 milk cows, respectively . All VTEC strains from the piglets produced only VT2vp, while the strains from the healthy pigs did not produce VT2vp, but did VT1 and/or VT2 . Most VTEC strains from calves and cows produced VT2vhb and some produced VT2 and VT1 . Serotyping of the isolated strains showed that many strains from the piglets belonged either O139:H1, O141:H4 or O141:HUT, but the strains from the pigs were either R-form or O-untypable . Many strains from the calves and cows were serotyped into O116 or O113, but there were several R-form and O-untypable . From these results, it is suggested that VTEC strains, especially from the pregnant pigs, calves with diarrhea and healthy milk cows, which produced the same type of Verotoxins to that produced by human isolates, may become sources of human infections.

J Biochem (Tokyo), 1992 Jul, 112(1), 45 - 51
Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor; Chen XM et al.; The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically . The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue . After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained . The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one . The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae . In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis . The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream . Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.

Genetika, 1992 Jul, 28(7), 186 - 8
{Construction of linear plasmid vectors for cloning in Escherichia coli cells}; Vostrov AA et al.; Prophage of the temperate coliphage N15 is a linear plasmid with covalently closed ends . The central part of plasmid N15 genome responsible for vegetative phage growth was replaced by DNA fragments containing genes for selective markers which have unique restriction sites . As a result a family of linear plasmid vectors was constructed . Their size is about 20 kb and their capacity is comparable with that of cosmid vectors.

Can J Vet Res, 1992 Jul, 56(3), 220 - 5
Epidemiological studies of congo red Escherichia coli in broiler chickens; Stebbins ME et al.; This prospective cohort study was designed to confirm the association between Congo red binding Escherichia coli (CREC) and E . coli air sacculitis in commercial broilers . It was also designed to evaluate CREC as an air sacculitis risk factor and to explore the CREC relationship to other air sacculitis risk factors (poultry house temperature, air-ammonia levels, and presence of other diseases) . In addition, this study was used to assess a possible role of the broiler-breeder flocks and hatchers in the spread of CREC air sacculitis . Congo red E . coli-associated airsacculitis risk was based on CREC exposure of the chicks in the hatchers . Breeder flocks with greater than 30 CREC colonies/plate from hatcher air sampling tests were placed in the high risk group; flocks with less than five CREC colonies/plate were placed in the low risk group . Increased risks of death due to air sacculitis (RR = 2.26), and increased death rates due to CREC air sacculitis (RR = 9.45) in high-risk flocks, identified CREC as an important air sacculitis risk factor . The attributable risk percent of CREC airsacculitis from hatcher exposure of CREC was 89.4%, pointing to the hatcher as the source of CREC infection . The association of specific broiler-breeder flocks to high levels of CREC in the hatchers, and subsequent air sacculitis, suggests that the broiler-breeders are the ultimate source of CREC.

Eur Cytokine Netw, 1992 Jul-Aug, 3(4), 417 - 20
Inhibition of morphine dependence by a lipopolysaccharide from Pantoea agglomerans; Okutomi T et al.; A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified and examined for relief of morphine dependence by observing its inhibition of the jumping of mice on naloxone-precipitate withdrawal . Administration of LPSp either intravenously or intradermally showed marked inhibition of the jumping . Beta-endorphin in mouse serum and brain tissue were recognized to be in synchrony with the time course of the relief . Administration of TNF-alpha gave similar effect, suggesting that LPSp induces a cytokine cascade to produce endogenous TNF followed by ACTH/beta-LPH gene products and beta-endorphin . The effect of LPSp was better than that of LPS from E . coli or Bordetella pertussis, and thus is considered to be applicable for clinical use.

Biophys J, 1992 Jul, 63(1), 28 - 34
Study of mechanisms of electric field-induced DNA transfection . III . Electric parameters and other conditions for effective transfection; Xie TD et al.; Electric parameters, osmolality, temperature, and pH of the suspending medium and the growth phase of cells, etc., are known to influence the efficiency of the pulsed electric field (PEF)-induced DNA transfection of cells . PEF-induced transfection of Escherichia coli JM105 by plasmid DNA PUC18, PUC19, PBR322, and PMSG has been used as a model system to establish quantitative relationships between these parameters and transfection efficiency . The main findings are summarized for experiments using unipolar square wave PEF . (a) For a given field strength (up to 6 kV/cm), the transfection efficiency (TE) was linearly dependent on the pulse width (up to 1 ms) . (b) When field strength is fixed, Log {TE} correlated with the number of pulses applied . Similarly, when field duration was fixed, Log {TE} correlated with the number of pulses . (c) In the absence of MgCl2, TE showed a maximal value at 50 mM sucrose and was reduced by several fold at lower and higher sucrose concentrations . Cell survival was nearly constant in the range 1-300 mM sucrose . (d) E . coli in the early and mid-exponential growth phases was more susceptible to PEF for DNA transfection than it was in the stationary phase . (e) For a given set of electric parameters, TE was the highest at neutral pH and was greatly reduced at acidic and alkaline pH . (f) Increasing the temperature from 0 to 37 degrees C resulted in the reduction of TE by three orders of magnitude . This could reflect a rapid shrinking of pores at higher temperatures . (g) TE was inversely proportional to the square of the size of the plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian Dis, 1992 Jul-Sep, 36(3), 504 - 11
Virulence factors of Escherichia coli associated with colisepticemia in chickens and turkeys; Emery DA et al.; Four hundred twenty turkey and 80 chicken Escherichia coli isolates from colisepticemic birds were examined for the following properties: heat-labile toxin (LT), heat-stable enterotoxin, verotoxin, colicinogenicity, hemolysin, and hydroxamate/aerobactin production . Twenty-four (5.7%) of the 420 turkey isolates and six (7.5%) of the 80 chicken isolates produced an LT that was cytotoxic for both Vero and Y-1 cells . In contrast, 48 (11.4%) of the turkey isolates and 18 (22.5%) of the chicken isolates produced a distinct LT that was cytotoxic only for Vero cells . In addition, 64 (80.0%) of the chicken isolates and 309 (74.0%) of the turkey isolates produced aerobactin . Colicinogenicity occurred in 51 (64.0%) of the chicken isolates, with 41 (51.0%) producing colicin V . By contrast, 254 (61.0%) of the turkey isolates produced a colicin, of which 176 (42.0%) produced colicin V . None of the chicken and turkey isolates produced hemolysin or heat-stable enterotoxin.

Ultramicroscopy, 1992 Jul, 42-44 ( Pt B), 1101 - 6
Imaging isolated strands of DNA molecules by atomic force microscopy; Thundat T et al.; We have employed an atomic force microscope (AFM) to image in air isolated strands of pBS+ plasmid DNA adsorbed onto freshly cleaved mica . At a DNA concentration below 0.3 micrograms/ml isolated strands of the plasmid DNA are usually seen, while for concentrations higher than 3 micrograms/ml a uniform coverage of interconnected DNA strands was observed . We found that the contrast and the width of DNA were dependent upon humidity . When the relative humidity exceeds 60%, negative contrast images with strand widths 20 times the width of DNA are found, while positive contrast images with 7 to 10 times the width of DNA are found when the humidity is below 30% . By placing the AFM in an environment where the humidity could be controlled, we were able to switch between positive and negative contrasts.

Radiobiologiia, 1992 Jul-Aug, 32(4), 560 - 5
{Changes in cellular radiosensitivity by modifying the cytoplasmic membranes}; Iarova MS et al.; The radiosensitivity of mouse myeloma and E . coli cells in the presence of Mg2+ and UO2(2+) ions has been investigated . It has been shown that Mg2+ ions (10(-4) M) do not influence the viability of E . coli and mouse myeloma cells . The presence of Mg2+ ions during irradiation reduces the survival rate of E . coli cells, but the addition of Mg2+ ions after irradiation does not influence the radiosensitivity of E . coli cells . Comparison of the results on the influence of Mg2+ ions upon cells and bilayer lipid membranes (BLM) permits us to suppose that Mg2+ ions increase the positive charge of the membranes thus promoting the increase in the number of short-lived radiolysis products which impair membranes and increase cell radiosensitivity . UO2(2+) ions (10(-4) M) increase the radioresistance of E . coli cells which can be associated with the increase in the lateral membrane viscosity, as it was shown in the studies on BLM.

Naunyn Schmiedebergs Arch Pharmacol, 1992 Jul, 346(1), 108 - 13
Pharmacokinetic and thrombolytic properties of unglycosylated recombinant tissue-type plasminogen activator (BM 06.021) produced in Escherichia coli; Martin U et al.; Recombinant tissue-type plasminogen activator (rt-PA) was produced in Escherichia coli cells in order to obtain an unglycosylated rt-PA (BM 06.021) with increased thrombolytic potency due to altered pharmacokinetic properties . The pharmacokinetics were studied in rabbits upon intravenous infusion of 200 kU/kg over 30 min . The thrombolytic dose-response effects were evaluated in a rabbit model with 125I-labeled venous thrombi upon intravenous infusion over 4 h . The thrombolytic effects after intravenous bolus injection of 200 kU/kg BM 06.021 were investigated in a canine model of coronary artery thrombosis . All studies were performed comparing BM 06.021 with glycosylated rt-PA (alteplase) . BM 06.021 demonstrated a longer (p less than 0.05) half-life (5.6 +/- 2.6 vs . 2.1 +/- 0.3 min) and a lower (p less than 0.05) clearance rate (7.5 +/- 0.8 vs . 22.2 +/- 3.1 ml.min-1.kg-1) than alteplase in rabbits upon intravenous infusion . The dose-response curve of BM 06.021 for thrombolysis in a rabbit model of jugular vein thrombosis was located to the left of that for alteplase with a 2.1-fold lower effective dose of 50% thrombolysis (ED50) of BM 06.021 (207 vs . 436 kU/kg) . Intravenous bolus injection of 200 kU/kg BM 06.021 induced the same reperfusion rate (4/6) as intravenous infusion of 800 kU/kg alteplase over 90 min in a canine model of coronary artery thrombosis . The residual thrombus wet weight did not significantly differ between BM 06.021 and alteplase (5.7 +/- 1.8 vs . 6.3 +/- 1.1 mg) . The results indicate that unglycosylated rt-PA (BM 06.021) has a higher in vivo thrombolytic potency than glycosylated rt-PA (alteplase).(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Epidemiol, 1992 Jul, 8(4), 548 - 52
Enterotoxigenic and necrotizing Escherichia coli in human diarrhoea in Spain; Blanco J et al.; Enterotoxigenic Escherichia coli (ETEC) strains of serotype 0153: K-:H45 CFA/I+ STa+ were associated with two outbreaks of neonatal diarrhoea that occurred in two different hospitals of Madrid, in one of which several children died . Two other outbreaks were associated with ETEC strains of serotypes 0159: K-:H21 (LT+) and 0159: K-:H4 (LT+ STa+) without CFA/I and CFA/II colonization factors . Necrotizing E . coli (NTEC) strains of serotype 06:K13, producing the cytotoxic necrotizing factor CNF1 and alpha-haemolysin, were also associated with two outbreaks of neonatal diarrhoea that occurred in a hospital in Madrid and in a hospital in Talavera de la Reina . The results of the characterization of some ETEC and NTEC strains isolated from sporadic cases of diarrhoea are also discussed.

Eur J Epidemiol, 1992 Jul, 8(4), 543 - 7
Heterogeneity among heat-labile enterotoxins produced by porcine enterotoxigenic Escherichia coli; Tsuji T et al.; The heat-labile enterotoxins (LT) produced by various porcine strains (LTp) of enterotoxigenic Escherichia coli were purified to homogeneity and their molecular properties were compared with each other . By double gel diffusion and rabbit skin permeability test, LTps from WT-1 (LTp-WT-1) and BO-149 (LTp-BO-149) strains were antigenically and biologically identical . By polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS), the mobilities of A and B subunits of LTp (BO-149) were identical to those of LTp (WT-1) . However, on polyacrylamide gel electrophoresis without SDS, LTp (BO-149) and LTp (WT-1) differed in mobility, suggesting their molecular differences . Though the pI value of the B subunit of LTp (BO-149) was identical to that of LTp (WT-1), the pI value of the A subunit of LTp (BO-149) was higher than that of LTp (WT-1) . These data suggest that there is molecular heterogeneity among LTps produced by the two porcine enterotoxigenic Escherichia coli strains.

Can J Microbiol, 1992 Jul, 38(7), 747 - 52
Virulence determinants of Escherichia coli: present knowledge and questions; Smith H; This paper describes the present state of research on the pathogenicity of Escherichia coli and points out the gaps in knowledge that should be filled in the future . First, the great versatility of E . coli in producing disease is noted, as well as the invaluable contributions that studies of it have made to the development of general knowledge on bacterial pathogenicity . Then, the biological requirements for pathogenicity: infection of mucous surfaces; penetration of those surfaces; multiplication in vivo; interference with host defence mechanisms; and damage to the host, are taken in turn, and an enquiry is made on how far studies have progressed toward identifying their molecular determinants and relating structure to biological action . Only for mucous surface adhesins and protein toxins are studies at the structure-function level . Some progress has been made on interference with host defence, but little is known about competition with commensals on mucous surfaces, invasion into the tissues, and growth in vivo.

Can J Microbiol, 1992 Jul, 38(7), 734 - 46
Escherichia coli cytotoxins and enterotoxins; Gyles CL; Vero cell cytotoxins and cytotonic enterotoxins produced by E . coli are toxic proteins, which have been implicated in a number of specific diseases in humans and animals . Nomenclature for these toxins is complicated by the existence of different names for the same toxin . The Vero cell cytotoxins are called verotoxins because they are lethal for Vero cells in culture; they are also known as Shiga-like toxins (SLTs) because they are clearly related to Shiga toxin in structure, amino acid sequence, mechanism of action, and biological activity . SLTs belong to two classes . SLT-I is identical with Shiga toxin and is in a class by itself (class I) . The other SLTs are closely related to each other and form a second class (class II) . Class II SLTs include SLT-II, SLT-IIv, SLT-IIvha, SLT-IIvhb, and SLT-IIva . All SLTs that have been investigated are A-B subunit protein toxins, whose A subunits possess N-glycosidase activity against 28S rRNA and cause inhibition of protein synthesis in eukaryotic cells . These toxins are enterotoxic as well as cytotoxic . SLTs produced in the intestine are absorbed into the blood stream and affect vascular endothelial cells in target organs . They may also have a direct toxic effect on enterocytes . Diseases in which E . coli SLTs have been implicated include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans and edema disease in pigs . Variation in receptor specificities among SLTs may be the reason for different disease syndromes in different host species . The E . coli enterotoxins belong to three distinct classes: heat-labile enterotoxin (LT), heat-stable enterotoxin type I or type a (STI, STa), and heat-stable enterotoxin type II or type b (STII, STb) . There is clear evidence that these cytotonic enterotoxins play an essential role in diarrheal disease . LT is an A-B subunit protein toxin, closely related to cholera toxin . Following binding of LT to receptors in enterocytes the A subunit is internalized . The enzymatically active A subunit transfers ADP-ribose from NAD to a GTP-dependent adenylate cyclase regulatory protein, thereby elevating intracellular levels of adenylate cyclase . The increased levels of cyclic AMP cause stimulation of A kinase and lead to hypersecretion of electrolytes and fluid . STI is a small peptide of 18 or 19 amino acids . It binds to receptors in enterocytes and stimulates particulate guanyl cyclase . Elevated intracellular cyclic GMP stimulates G kinase, resulting in increased Cl- secretion and impaired absorption of Na+Cl- . STII is a peptide toxin whose mechanism of action is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Can J Microbiol, 1992 Jul, 38(7), 728 - 33
Mechanism and regulation of synthesis of aerobactin in Escherichia coli K12 (pColV-K30); Neilands JB; The aerobactin operon of the virulence plasmid pColV-K30 of Escherichia coli K12 consists of four genes for biosynthesis and one for transport of the siderophore . Regulation by iron occurs at the transcriptional level and is mediated by a ferrous iron binding protein designated Fur (ferric uptake regulation) . The metallated Fur repressor binds at a palindromic dyad, the "iron box" operator, situated in the vicinity of the RNA polymerase attachment site of the promoter . Evidence suggests that the ferrous iron enters the C-terminal domain of Fur to cause a conformational change in the N-terminal part of the protein . This results in greatly enhanced affinity of the repressor for the operator.

Hum Antibodies Hybridomas, 1992 Jul, 3(3), 137 - 45
Preparation of genetically engineered monoclonal antibodies for human immunotherapy; Parren PW; Production of human monoclonal antibodies (MAbs) of predefined specificity using conventional hybridoma technology has proved to be difficult . Immunotherapeutic intervention in humans with rodent MAbs, however, is disadvantageous because of the antigenicity of these proteins and may result in human antibody responses against this foreign agent . To circumvent this problem, recombinant DNA techniques have been developed to transplant the specificity of a rodent MAb into a human antibody . Two basic approaches are being employed: first, combining rodent MAb variable regions with human constant regions; and more recently, "reshaping" human MAbs by grafting complementarity-determining regions (CDR) into the human antibody framework . These humanized MAbs can now be used to study human Fc-dependent effector mechanisms in detail, which seems essential to optimize potential in vivo application.

Hum Antibodies Hybridomas, 1992 Jul, 3(3), 129 - 36
Stable expression of cloned human antibody genes in murine myeloma cells; Knight DM et al.; Human monoclonal antibodies (MAbs) offer potential advantages over murine MAbs for therapy because they are not likely to elicit immune responses and are expected to interact more efficiently with the human immune system to activate therapeutically useful functions . Traditional methods for obtaining human MAbs (i.e., immortalization of B cells by cell fusion or transformation) can result in low and unstable antibody secretion . Recently, methods have been devised for direct cloning of human variable region genes via polymerase chain reaction and phage combinatorial libraries . Both types of human MAb production can benefit from expression systems that support the stable, high-level antibody secretion required for therapeutic use . Using an existing human-derived hybridoma that secretes a human IgM antibody as a convenient source of antibody genes, we have demonstrated that cloned human antibody genes can be efficiently expressed in murine myeloma cells and that cell lines with properties suitable for large-scale economical production can be obtained . We were unable to detect any differences between the antibodies produced by the original hybridoma and the engineered cell line . In addition, we were able to express an IgG form of the antibody, showing that expression of a recombinant human antibody need not be limited to the original antibody class.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5705 - 9
Cloning, expression, and crystallization of recoverin, a calcium sensor in vision; Ray S et al.; Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range . We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library . The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter . A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture) . The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97) . Myristoylated recombinant recoverin formed in this way in E . coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum . The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies . The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A . These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A . The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

Gene, 1992 Jul 1, 116(1), 81 - 6
Construction and expression of diphtheria toxin-encoding gene derivatives in Escherichia coli; Zdanovsky AG et al.; We reported earlier that in the periplasmic space of Escherichia coli, truncated derivatives of diphtheria toxin undergo limited proteolysis {Zdanovsky et al., Mol . Biol . 22 (1988) 1037-1293} . Here, we present data indicating that this proteolysis is reduced in cells bearing a mutation in the degP gene . We have also constructed hybrid genes whose products are not secreted into the periplasm . These hybrid genes were expressed in E . coli from both the pR promoter, controlled by the heat-inducible CI857 repressor, and from the P(lac) promoter, controlled by the IPTG-inducible LacI repressor . The latter system proved to be more productive.

J Med Microbiol, 1992 Jul, 37(1), 15 - 21
The iron uptake mechanisms of enteropathogenic Escherichia coli: the use of haem and haemoglobin during growth in an iron-limited environment; Law D et al.; The iron uptake mechanisms of enteropathogenic Escherichia coli (EPEC) were examined and compared with those of control E . coli strains . The incidence of aerobactin production was similar (39% and 37% respectively) in the two groups . The quantities of enterochelin produced by aerobactin-negative EPEC and control strains were similar, as were the quantities of enterochelin produced by aerobactin-positive EPEC and control strains . The ability to use haem or haemoglobin as an iron source in an iron-restricted environment was found in 80.4% and 60.8% of EPEC strains respectively, and in 76.6% and 56.6% of control E . coli strains . The ability of E . coli strains to use these compounds was not related to the production of enterochelin or aerobactin or to the production of haemolysins, and may be an important characteristic of bowel organisms . When growing in an iron-limited environment, the iron contained in haemoglobin was used in preference to ovotransferrin-bound iron . During periods of haemoglobin-stimulated growth, the enterochelin uptake system was shown to be fully expressed and may be involved in transport of haemoglobin-derived iron into the cell . Uptake of ovotransferrin-bound iron took place immediately upon exhaustion of haemoglobin-derived iron . The ability to use iron derived from haem compounds represents an alternative iron uptake mechanism for organisms growing in an iron-limited environment and allows greater flexibility during growth in vivo.

J Bacteriol, 1992 Jul, 174(14), 4769 - 76
Regulation of katF and katE in Escherichia coli K-12 by weak acids; Schellhorn HE et al.; Chromosomal transcriptional and translational lacZ fusions to the katE (structural gene for the HPII hydroperoxidase) and katF (putative sigma factor required for katE expression) genes of Escherichia coli were isolated, and the regulation of these fusions was used to identify factors that control the expression of these two important antioxidant factors . While katE was found to be regulated primarily at the level of transcription (since induction patterns were similar for both transcriptional and translational fusions), katF expression was a function of both transcriptional and translational signals . The katE gene was induced 57-fold as cells entered the stationary phase, while katF was induced 23-fold . katF induction was coincident with katE induction and occurred at the onset of the stationary growth phase . Expression of both katE and katF could be induced by resuspending uninduced exponential-phase cells in spent culture supernatant recovered from stationary-phase cells . The component of stationary-phase culture supernatant responsible for induction of the katF regulon appeared to be acetate, since expression of both katE and katF fusions was induced when exponential-phase cells were exposed to this weak acid . Other weak acids, including propionate and benzoate, were also found to be effective inducers of expression of both katF and katE . Induction of katE and katF fusions was unaffected in merodiploid strains containing both mutant and wild-type alleles, indicating that expression of both genes is independent of the wild-type gene product . Examination of catalase zymograms prepared from cells exposed to various levels of acetate revealed that both HPI and HPII catalases are induced by this weak acid, suggesting that there is a common link in the regulation of these two enzymes.

J Bacteriol, 1992 Jul, 174(13), 4496 - 9
Mutational analysis of the glycine-rich region of the c subunit of the Escherichia coli F0F1 ATPase; Norris U et al.; Eight strains carrying amino acid substitutions within the c subunit of the F0F1 ATPase of Escherichia coli have been constructed by using site-directed mutagenesis . Three strains carrying the substitutions Gly-23----Leu, Ala-24----Leu, and Gly-38----Leu, which reside in or near the highly conserved glycine-rich region of the c subunit, are unable to carry out oxidative phosphorylation . Membranes prepared from these strains possess basal levels of ATPase activity . In contrast, strains carrying the substitutions Ile-30----Phe, Gly-33----Leu, Gly-58----Leu, and Lys-34----Val and the Lys-34----Val, Glu-37----Gln double substitution were found to possess a coupled phenotype similar to that of the wild type.




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