Escherichia coli

Biochemistry, 1992 Jan 14, 31(1), 32 - 9
Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase . Preparation and properties of a cysteine-free enzyme; Gloss LM et al.; The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A) . Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S) . A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed . The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation . The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry . The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT . The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea . Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C . Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential . The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines . We consider the evolutionary implications of these findings.

Biochemistry, 1992 Jan 14, 31(1), 155 - 62
Serine hydroxymethyltransferase: origin of substrate specificity; Angelaccio S et al.; All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate . For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue . The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties . The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme . The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme . The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme . The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme . The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm . Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process . This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jan 14, 31(1), 106 - 10
Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization; Boiteux S et al.; We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) {Boiteux, S., O'Connor, T . R., Lederer, F., Gouyette, A., & Laval, J . (1990) J . Biol . Chem . 265, 3916-3922} . DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB) . The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples . Thirteen products resulting from pyrimidines and purines were detected in gamma-irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA . Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8-OH-Gua . The excision of a small but detectable amount of 8-hydroxyadenine was also observed . The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme . Nine pyrimidine-derived lesions were not excised . The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA . The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jan 14, 31(1), 133 - 8
Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism; Cho H et al.; The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates . A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme . A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide . Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1 . The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate . Brief incubation of all three PTPase fragments with a {32P}phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes . Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.

Biochemistry, 1992 Jan 14, 31(1), 121 - 32
Physical properties of the Escherichia coli transcription termination factor rho . 2 . Quaternary structure of the rho hexamer; Geiselmann J et al.; Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry {Geiselmann, J., Yager, T . D., Gill, S . C., Calmettes, P., & von Hippel, P . H . (1992) Biochemistry (preceding paper in this issue)} . Here we describe studies that further define the quaternary structure of this hexamer . We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface . Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry . Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer . Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry . The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry . We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer . Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.

Biochemistry, 1992 Jan 14, 31(1), 111 - 21
Physical properties of the Escherichia coli transcription termination factor rho . 1 . Association states and geometry of the rho hexamer; Geiselmann J et al.; To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex . To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers . Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment . The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance . Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A . The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A . These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy . In the following paper in this issue {Geiselmann, J., Seifried, S . E., Yager, T . D., Liang, C., & von Hippel, P . H . (1992)}, these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.

Biochemistry, 1992 Jan 14, 31(1), 22 - 6
Circular DNA molecules imaged in air by scanning force microscopy; Bustamante C et al.; Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished . Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities . In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained . This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus . Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity . The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.

Biochemistry, 1992 Jan 14, 31(1), 12 - 6
Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine; Hugues M et al.; Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography . The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose . Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix . Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts . Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations . Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa . Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit . In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors . Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M . These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors . The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Biotechniques . 1992 Jan;12(1):40, 42, 44.
A rapid PCR method of screening for small mutations; Major JG Jr; We report a modified method of screening for point mutations using a PCR approach based upon the sensitivity of PCR to the 3' terminus of the primer . This method provides a sensitive screen when using either plasmid DNA or bacterial cell lysates . We have optimized the technique for general use to allow rapid screening of mutants with good discrimination . Unlike previous similar methods, this technique has no inherent limitation in primer design on the 3'-terminal base chosen.

J Bacteriol, 1992 Jan, 174(2), 623 - 6
Molecular characterization of mutations affecting expression level and growth rate-dependent regulation of the Escherichia coli zwf gene; Rowley DL et al.; We characterized three cis dominant mutations which elevate glucose 6-phosphate dehydrogenase level . Growth rate-dependent regulation and oxidative stress control of enzyme level were altered by the mutations . DNA sequencing and transcript mapping showed that the "up" mutations created new promoters whose hyperactive expression overrides the normal regulation of the native promoter.

J Bacteriol, 1992 Jan, 174(1), 92 - 101
Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations; Puziss JW et al.; It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process . The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4 . This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0 . One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective . Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E . coli protein export machinery . One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects . Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E . coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide . In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666 . These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 23 - 30
Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene; Barletta RG et al.; Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (beta-galactosidase) gene which lacked a promoter, a ribosome binding site, and an ATG start codon . Libraries of mycobacteriophage Bxb1, L1 and TM4 DNAs were constructed, and introduced by electroporation into Mycobacterium smegmatis and the 'bacille Calmette-Guerin' (BCG) . Clones carrying mycobacterial expression sequences were detected by their blue colour or characteristic fluorescence when plated on media containing chromogenic or fluorogenic substrates . Varying degrees of beta-galactosidase expression were observed, and one Bxb1 expression signal was identified where beta-galactosidase expression is repressed in phage lysogens.

Prostate, 1992, 20(2), 113 - 6
Prostate specific antigen and prostatitis . II . PSA production and release kinetics in vitro; Moon TD et al.; Elevations in serum prostate specific antigen (PSA) levels in patients with prostatitis are well known, but the pathophysiologic mechanisms involved with this phenomenon are poorly understood . We have recently evaluated the effect of prostatitis on PSA levels in primates . This data demonstrated a rapid rise in PSA, which subsequently fell along the biological decay curve . This suggested a release of sequestered PSA . We evaluated the effect of infection upon PSA production for the prostatic adenocarcinoma cell line LNCaP . The cell line was determined to produce 9.6 +/- 2.7 fg/cell/hr PSA with essentially linear kinetics . No effect was seen with dead bacteria, bacterial supernatant or complement upon the PSA production . Live E . coli had no effect for 4-8 hours at which time the cells sloughed and PSA production ceased . No release of stored PSA was seen . These data do not elucidate the reasons for increased serum PSA levels found with acute bacterial prostatitis, but indicate that it is not a storage phenomenon.

J Ind Microbiol, 1992 Jan, 9(1), 1 - 9
Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinant Escherichia coli strains; Li X et al.; Four recombinant strains of Escherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth . Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding beta-galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved oxygen control . The biomass of all strains under constant aerobic conditions was 12-36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control . The plasmid content in all strains under aerobic conditions was 2.9-11.7 times higher than that under aerobic conditions . The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain . In no strain were constant aerobic conditions optimal . However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.

J Bacteriol, 1992 Jan, 174(2), 514 - 24
Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli; Wilson HR et al.; Expression of the pyrC gene, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, is negatively regulated by pyrimidine availability in Escherichia coli . To define the mechanism of this regulation, an essential regulatory region between the pyrC promoter and the initial codons of the pyrC structural gene was identified . Mutational analysis of this regulatory region showed that the formation of a hairpin at the 5' end of the pyrC transcript, which overlaps the pyrC ribosome binding site, is required for repression of pyrC expression . Formation of the hairpin appears to be controlled by nucleotide-sensitive selection of the site of pyrC transcriptional initiation . When the CTP level is high, the major pyrC transcript is initiated with this nucleotide at a position seven bases downstream of the pyrC -10 region . This transcript is capable of forming a stable hairpin at its 5' end . When the CTP level is low and the GTP level is high, conditions found in cells limited for pyrimidines, the major pyrC transcript is initiated with GTP at a position two bases further downstream . This shorter transcript appears to be unable to form a stable hairpin at its 5' end . These results suggest a model for regulation in which the longer pyrC transcripts are synthesized predominantly under conditions of pyrimidine excess and form the regulatory hairpin, which blocks pyrC translational initiation . In contrast, the shorter pyrC transcripts are synthesized primarily under conditions of pyrimidine limitation, and they are readily translated, resulting in a high level of dihydroorotase synthesis . The data also indicate that a low level of pyrimidine-mediated regulation may occur at the level of transcriptional initiation.

Acta Chir Hung, 1992-93, 33(1-2), 197 - 208
Protective effect of radio-detoxified endotoxin (Tolerin) on the ultrastructure of pancreas in experimental endotoxin shock of rats; Bende S Jr et al.; The ultrastructural changes of pancreas exocrine cells were studied after the intravenous administration of endotoxin (LPS) or radio-detoxified endotoxin (150 kGy 60Co-gamma irradiated: RD-LPS or Tolerin) . The LPS (1 mg/rat) induces an autolytic destruction in the membranes of the mitochondria of the pancreas exocrine cells . The RD-LPS given in similar dose does not produce any autolytic change . However, a small dose (100/micrograms/rat) of RD-LPS (Tolerin) as a pretreatment can protect the autolytic destruction of the mitochondria induced by LPS . This may be attributed to the membrane stabilizing effect of RD-LPS.

Res Microbiol, 1992 Jan, 143(1), 5 - 14
Effect of Bdellovibrio bacteriovorus infection on the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli: evidence for activation of cytoplasmic proteolysis; Romo AJ et al.; Intact cells of Bdellovibrio bacteriovorus strain 109J were found to be incapable of taking up 14C-methyl alpha-glucoside, mannitol or fructose, and extracts derived from these cells exhibited negligible activities of the protein components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . Escherichia coli strain ML35 cells exhibited high in vivo sugar uptake activities that were progressively lost over a period of 2 h at 30 degrees C following the entry of B . bacteriovorus into the periplasm of E . coli . In vitro complementation assays revealed that the E . coli PTS enzymes, enzyme I, HPr, and the glucose- and mannitol-specific enzymes II, were all lost almost in parallel with the disappearance of uptake activity . Thus, loss of activity in vivo was not due to membrane leakiness, energy depletion, or preferential inhibition or inactivation of any one protein component of the PTS . Instead, loss of PTS activity was attributed to digestion of the protein constituents of the system by proteases present in the cytoplasm of the host cell after bdellovibrio entry . Both ethylenediaminetetraacetate and phenylmethylsulphonyl fluoride partially protected against inactivation in vitro, and the two inhibitors together gave full protection, suggesting that both metallo- and seryl-proteases were responsible for the inactivation . Protease activity increased progressively with time following bdellovibrio entry and appeared to degrade the E . coli PTS enzymes in vivo . Preliminary evidence suggested that the proteases responsible for PTS enzyme degradation may be encoded by the B . bacteriovorus chromosome.

J Membr Biol, 1992 Jan, 125(1), 81 - 91
Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: a study using fluorescence measurements through polycarbonate supports; Akeson M et al.; We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV) . Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells . When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection . This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface . Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF . Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2 . Neither increase could explain the block of VSV infectivity by plasma membrane depolarization . One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.

Nucleic Acids Res, 1992 Jan 11, 20(1), 41 - 8
In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins; Scherzinger E et al.; We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT . In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment . Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently . The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB . The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010 . We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form . Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.

Nucleic Acids Res, 1992 Jan 11, 20(1), 105 - 9
Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction; Malki A et al.; The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences . In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction . Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template . We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle.

Cell, 1992 Jan 10, 68(1), 133 - 42
A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer; Smith DL et al.; DNA recognition sequences for dimeric proteins typically contain two types of information . The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites . We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type . Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats . We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators . These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.

Science, 1992 Jan 10, 255(5041), 203 - 6
Dimerization of a specific DNA-binding protein on the DNA; Kim B et al.; Many specific DNA-binding proteins bind to sites with dyad symmetry, and the bound form of the protein is a dimer . For some proteins, dimers form in solution and bind to DNA . LexA repressor of Escherichia coli has been used to test an alternative binding model in which two monomers bind sequentially . This model predicts that a repressor monomer should bind with high specificity to an isolated operator half-site . Monomer binding to a half-site was observed . A second monomer bound to an intact operator far more tightly than the first monomer; this cooperativity arose from protein-protein contacts.

Science, 1992 Jan 10, 255(5041), 197 - 200
Site-specific incorporation of novel backbone structures into proteins; Ellman JA et al.; A number of unnatural amino acids and amino acid analogs with modified backbone structures were substituted for alanine-82 in T4 lysozyme . Replacements included alpha,alpha-disubstituted amino acids, N-alkyl amino acids, and lactic acid, an isoelectronic analog of alanine . The effects of these electronic and structural perturbations on the stability of T4 lysozyme were determined . The relatively broad substrate specificity of the Escherichia coli protein biosynthetic machinery suggests that a wide range of backbone and side-chain substitutions can be introduced, allowing a more precise definition of the factors affecting protein stability.

Biochim Biophys Acta, 1992 Jan 9, 1118(2), 107 - 15
Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli; Kelley MJ et al.; Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids . A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter . In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene . Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies . The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea . The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT . After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+ . The renatured recombinant protein was purified by Affi-gel blue column chromatography . The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme . This is the first report of the expression of a recombinant PLA2 from any venom.

J Theor Biol, 1992 Jan 7, 154(1), 91 - 107
Phospholipid domains determine the spatial organization of the Escherichia coli cell cycle: the membrane tectonics model; Norris V; Escherichia coli normally divides at its equator between segregated nucleoids . Such division is inhibited during perturbations of chromosome replication (even in the absence of inducible division inhibitors); eventually, division resumes at sites which are not at this equator . Escherichia coli will also divide at its poles to generate minicells following overproduction of the FtsZ or MinE proteins . The mechanisms underlying the division inhibition and the positioning of the division sites are unknown . In the membrane tectonics model, I propose that the formation of phospholipid domains within the cytoplasmic membrane positions division sites . The particular phospholipid composition of a domain attracts particular proteins and determines their activity; conversely, particular proteins change the composition of domains . Principally via such proteins, the interaction of the chromosome with the membrane creates a chromosomal domain . The development of chromosomal domains during replication and nucleoid formation contributes to the formation and positioning of a septal domain between them . During septation (cell division), this septal domain matures into a polar domain . Each domain attracts and activates different enzymes . The septal domain attracts and activates enzymes necessary for septation . Preventing the formation of the septal domain by preventing chromosome replication prevents normal division . Altering the composition of the polar domain may allow septation enzymes to function there and generate minicells . A corollary of the model explains how the formation of an origin domain by the attachment of hemi-methylated origin DNA to the membrane may underlie the creation and migration of structures within the envelope, the periseptal annuli.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 177 - 82
Selective inhibition of the polypeptide chain elongation in eukaryotic cells; Tujebajeva RM et al.; The effect of Cephalotaxus alkaloids--homoharringtonine and cephalotaxine--on translation in a cell-free system from rabbit reticulocytes and on phenylalanine polymerisation by human ribosomes was studied . The effect of the alkaloids on the nonenzymatic and the eEF-1-dependent Phe-tRNA(Phe) binding to poly(U)-programmed 80S ribosomes, diphenylalanine synthesis accompanying nonenzymatic Phe-tRNA(Phe) binding and acetylphenylalanyl-puromycin formation was examined . Homoharringtonine was shown to inhibit the formation of diphenylalanine and acetylphenylalanyl-puromycin catalysed by human and rat liver ribosomes, but was inactive as an inhibitor on the E . coli elongation system . Neither nonenzymatic nor enzymatic Phe-tRNA(Phe) binding was noticeably affected by the alkaloid . It has been proposed that the site of homoharringtonine binding to 80S ribosomes should overlap or coincide with the acceptor site of the ribosomal peptidyl transferase centre . The association constant of homoharringtonine for 80S human ribosomes was estimated to be (2.57 +/- 0.33).10(7) M-1 in the presence of puromycin . Cephalotaxine did not exert a significant influence on the polypeptide chain elongation.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 161 - 5
Enhancement of DNA transfection efficiency by heat treatment of cultured mammalian cells; Takai T et al.; The expression of genes introduced into various mammalian cell lines was enhanced by raising the temperature of the cells to 42 degrees C for a few hours after DNA transfection . This heat treatment resulted in an up to 10-fold increase in the frequency of the cells that transiently expressed a foreign gene such as that of beta-galactosidase, whereas it had only a limited enhancing effect on the development of stable transformants . By immunotitration analysis, it was confirmed that the enhanced expression of beta-galactosidase activity correlated well with the increase of the enzyme protein . This procedure may have an applicability for augmenting the frequency of transient gene expression in many cell types.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 149 - 54
Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme; Lundstrom K et al.; To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14 . Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside . Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting . Both enzymes were purified from E . coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 145 - 8
An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3?
Nagel K, Voigt J.
An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-{35S}Met-tRNAfMet to ribosomes . In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-{35S}Met-tRNAfMET/poly(A,U,G)/ribosome complexes . Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet . Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3 . The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor . Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits.

FASEB J, 1992 Jan 6, 6(2), 759 - 64
High-level expression of functional human cytochrome P450 1A2 in Escherichia coli; Fisher CW et al.; Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA . The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21 . The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein . E . coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 219 - 22
The 16S rRNA gene of Streptomyces lividans TK64 contains internal promoters; Chang SC et al.; A 632-bp Sau3AI fragment of Streptomyces lividans TK64 genome was found to confer promoter activity in Streptomyces and Escherichia coli . This fragment showed almost identical sequence (97.8%) to the S . coelicolor 16S rRNA segment encompassing from nucleotide 706 to 1337 region . The transcription start points of this fragment were identified by the primer extension method . Analysis of the nucleotide sequence upstream the transcription start points revealed two putative E . coli-like promoters resided within this fragment . The occurrence of internal promoters active in Streptomyces and E . coli was also confirmed in the 16S rRNA gene of rrnE operon from TK64.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 172 - 6
Induction by psychotropic drugs and local anesthetics of DnaK and GroEL proteins in Escherichia coli; Tanji K et al.; We examined effects of psychotropic drugs and local anesthetics on the synthesis of heat shock proteins in Escherichia coli . Chlorpromazine, a phenothiazine derivative, was shown to induce DnaK and GroEL proteins, major heat shock proteins in E . coli . The inductions of these proteins were not observed in an rpoH (= htpR) amber mutant strain, indicating that the heat shock sigma factor sigma 32 was required for their inductions . Northern blot hybridization analysis revealed that chlorpromazine induced increases of messenger RNAs for the DnaK and GroEL proteins . Thus, the induction occurred at the level of transcription . Chlorpromazine also induced non-heat shock proteins with molecular masses of 21 kDa, 20 kDa, and 17 kDa, even in the rpoH mutant strain . Other psychotropic drugs and local anesthetics, namely, dibucaine, lidocaine, imipramine, tetracaine and procaine, also induced DnaK and GroEL proteins and the small molecular weight proteins.

Biochim Biophys Acta, 1992 Jan 6, 1129(2), 215 - 8
Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli; Wilson VG et al.; Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli . Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame) . The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s).

J Mol Biol, 1992 Jan 5, 223(1), 31 - 40
Leftward ribosome frameshifting at a hungry codon; Gallant JA et al.; Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames . Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply . Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.

J Mol Biol, 1992 Jan 5, 223(1), 159 - 70
Expectation maximization algorithm for identifying protein-binding sites with variable lengths from unaligned DNA fragments; Cardon LR et al.; An Expectation Maximization algorithm for identification of DNA binding sites is presented . The approach predicts the location of binding regions while allowing variable length spacers within the sites . In addition to predicting the most likely spacer length for a set of DNA fragments, the method identifies individual sites that differ in spacer size . No alignment of DNA sequences is necessary . The method is illustrated by application to 231 Escherichia coli DNA fragments known to contain promoters with variable spacings between their consensus regions . Maximum-likelihood tests of the differences between the spacing classes indicate that the consensus regions of the spacing classes are not distinct . Further tests suggest that several positions within the spacing region may contribute to promoter specificity.

J Mol Biol, 1992 Jan 5, 223(1), 131 - 44
Direct evidence for the effect of transcription on local DNA supercoiling in vivo; Rahmouni AR et al.; The B-to-Z structural transition of varying lengths (74 to 14 base-pairs) of (CG) tracts has been used as a superhelicity probe to examine the local topological changes induced by transcription at defined genetic loci in vivo . The local-topology reporter sequences indicate that under steady-state transcription the region upstream from the promoter experiences an increase in negative supercoiling whereas the region downstream from the terminator displays a decrease in negative superhelicity . This result provides direct in vivo evidence for the notion that the translocation of an RNA polymerase elongation complex along the double-helical DNA generates positive supercoils in front of it and negative supercoils behind it . Also, this twin-supercoiled domain model was tested inside a transcribed region where a high degree of negative supercoiling generated by the passage of each individual RNA polymerase was detected . Hence, these data indicate that the induced supercoils are confined to the vicinity of each RNA polymerase complex in a multipolymerase system.

J Mol Biol, 1992 Jan 5, 223(1), 115 - 29
C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs; Tateishi S et al.; RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end . The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA . Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein . A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein . Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind . The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein . The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein . The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed.

J Mol Biol, 1992 Jan 5, 223(1), 105 - 14
Inhibitory effects of N- and C-terminal truncated Escherichia coli recA gene products on functions of the wild-type recA gene; Horii T et al.; The effects of the expression of Escherichia coli truncated RecA protein on the host recA functions were examined . The recA gene on a multicopy plasmid was manipulated to express the truncated RecA protein from its carboxyl (C) and amino (N) terminal ends where a maximum of four extra amino acid residues was added . The regulatory part of the recA gene was substituted by the lacUV5 promoter in the plasmid to facilitate the artificial control of recA expression . Enzyme-linked immunosorbent assay and Western blot analyses revealed great differences in accumulation of the truncated RecA proteins in the cell, depending on the location of the site of truncation . The expression of truncated proteins lacking 62, 77, 93 or 149 amino acid residues from the C-terminal end caused the host recA+ wild-type cell to become sensitive to ultraviolet irradiation and interfered with chromosomal recombination but did not interfere with the induction of lambda prophage . The expression of truncated RecA protein with 25 amino acid residues deleted from the C-terminal end caused the host cell to induce SOS functions constitutively . Truncated RecA proteins with 15 or 28 amino acid residues missing from the N-terminal end severely interfered with all of the host recA functions examined here . The effect of the loss of 41 amino acid residues from the N-terminal end of RecA was significant but less than the effect of proteins lacking 15 or 28 amino acid residues from the N-terminal end . A protein lacking 59 amino acid residues from the N-terminal end showed little interference with any measured recA functions, suggesting that the deletion of the region from around residues 41 to 59, which is rich in hydrophobic side-chains, influenced the ability of the truncated protein to interfere with the functions of wild-type RecA protein . We also constructed a mutant gene with an internal deletion whose product was missing a region from residues 184 to 204 . That mutant RecA protein was stably accumulated in the cell . This protein had little effect on the function of host wild-type recA gene product . The possible function of the regions at the N and C termini are discussed.

J Biol Chem, 1992 Jan 5, 267(1), 91 - 5
Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli; Ko YH et al.; Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli . Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation . Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate . Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity . The photomodified enzyme was labeled by reduction with NaB{3H}4 in the presence and absence of the substrates succinate plus glyoxylate . Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase . These residues are highly conserved in all five known sequences of this enzyme.

J Biol Chem, 1992 Jan 5, 267(1), 542 - 5
GTPase-mediated activation of ATP sulfurylase; Leyh TS et al.; GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism . The activation of the enzyme, purified from Escherichia coli, is titratable with GTP . The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP . The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis . The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins . This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase.

J Biol Chem, 1992 Jan 5, 267(1), 419 - 25
(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin; Heath WF et al.; The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide . Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin . A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain . To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free . Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein . By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity . Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding . Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin . The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin . Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation . The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.

J Biol Chem, 1992 Jan 5, 267(1), 35 - 8
Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation . In vitro dephosphorylation of UBF reduces its transactivation properties; O'Mahony DJ et al.; We have analyzed the expression, phosphorylation, and localization of the ribosomal DNA transcription factors UBF1 and UBF2 in Chinese hamster ovary cells in response to serum deprivation . In vivo labeling experiments demonstrate that UBF1 and UBF2 are phosphoproteins . Phosphoamino acid analysis of the in vivo labeled proteins demonstrate that UBF is phosphorylated on serine residues . Following serum deprivation there is no alteration in the cellular levels of UBF1 and UBF2 as determined by Western blotting, but there is an 80% reduction in the level of phosphorylation of UBF compared with logarithmically growing cells . Following serum deprivation there is a redistribution of UBF between the nucleolus, the nucleus, and the cytoplasm . Phosphatase-treated UBF demonstrated a reduced ability to rescue transcription by RNA polymerase I from the rDNA spacer promoter in vitro . These findings suggest that phosphorylation of UBF is a prerequisite for transactivation of RNA polymerase I.

J Biol Chem, 1992 Jan 5, 267(1), 150 - 8
Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine . Characterization of sequences at the inhibitor and coenzyme binding sites; Poulin R et al.; Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO) . The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB{3H}4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing . This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes . The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E . coli . Using a similar procedure to analyze ODC labeled by reaction with {5-14C}DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor . Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs . These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC . Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine . This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction . This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.

J Mol Biol, 1992 Jan 5, 223(1), 79 - 93
RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA; Chow SA et al.; In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament . Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules . The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule . However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end . Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products . By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange . The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure . We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand . The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.

J Mol Biol, 1992 Jan 5, 223(1), 9 - 15
Bar to normal UGA translation by the selenocysteine tRNA; Li WQ et al.; The selC gene product, tRNA(Sec), inserts selenocysteine at UGA (opal) codons in a specialized mRNA context . We have investigated the action of the tRNA at ordinary UGA codons, normally not translated, by changing the unusual structural features of tRNA(Sec) . Sequences in the D arm, CCA arm and variable arm of the tRNA all contribute to the prohibition against translation of ordinary UGA codons . One multiple mutant is a moderately efficient serine-inserting UGA suppressor tRNA.

J Mol Biol, 1992 Jan 5, 223(1), 41 - 54
Mechanism of post-segregational killing by the hok/sok system of plasmid R1 . Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene; Thisted T et al.; The hok/sok locus of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNAs, Hok mRNA and Sok antisense RNA . Hok mRNA encodes the Hok killer protein of 52 amino acid residues . Expression of hok is regulated post-transcriptionally by Sok antisense RNA . Killing of plasmid-free daughter-cells by the hok/sok system is accomplished through differential decay of the Hok and Sok-RNAs: Hok mRNA is very stable while Sok-RNA decays rapidly, thus leading to derepression of Hok mRNA translation in plasmid-free segregants, ensuring a rapid and selective killing of these cells . Sok antisense RNA is complementary to the leader region of the Hok mRNA . However, the region of complementarity does not overlap with the hok Shine-Dalgarno sequence . Thus, Sok-RNA regulates hok translation indirectly by an as yet unknown mechanism . We show here that Sok antisense RNA regulates the translation of another reading frame located in the hok/sok locus . This new reading frame, which overlaps with almost the entire hok gene, was denoted mok (mediation of killing) . Point-mutations that prevent mok translation through the hok translational initiation region abolish efficient expression of hok . Furthermore, these mutations abolish the Sok-RNA-mediated control of hok gene expression . Hence, the antisense-RNA-mediated regulation of the hok gene seems to occur via translational coupling between the hok and mok reading-frames.

J Biol Chem, 1992 Jan 5, 267(1), 144 - 9
A relationship between asparagine synthetase A and aspartyl tRNA synthetase; Hinchman SK et al.; A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A . The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase . To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases . The resulting genes were expressed in E . coli, and the gene products were assayed for asparagine synthetase activity in vitro . Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity . Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298 . Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity . These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E . coli, evolved from an ancestral aminoacyl tRNA synthetase.

J Biol Chem, 1992 Jan 5, 267(1), 526 - 41
Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase . An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility; Zhao GP et al.; The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation . The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281 . Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides . The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro . In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner . The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type . In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression . At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged . The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies . An open versus closed model of conformational change within the beta 2 protein is proposed . A plausible role for the hinge region is discussed.

J Biol Chem, 1992 Jan 5, 267(1), 413 - 8
In vitro translocation of secretory proteins possessing no charges at the mature domain takes place efficiently in a protonmotive force-dependent manner; Kato M et al.; The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied . Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein . The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force . Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins . The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain . All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size . The mature domain of the smallest one was 45 amino acid residues in length . Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.

Biochemistry, 1991 Dec 24, 30(51), 11788 - 95
Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain; Maeda-Yorita K et al.; The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197 . Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor . The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) {Russell, G . C., Allison, N., Williams, C . H., Jr., & Guest, J.R . (1989) Ann . N.Y . Acad . Sci . 573, 429-431} . Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase . Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase . Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase . The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence . The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase . On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level . Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1992 Jan 3, 255(5040), 85 - 7
Interaction of p107 with cyclin A independent of complex formation with viral oncoproteins; Ewen ME et al.; The p107 protein and the retinoblastoma protein (RB) both bind specifically to two viral oncoproteins, the SV40 T antigen (T) and adenoviral protein E1A (E1A) . Like RB, p107 contains a segment (the pocket) that, alone, can bind specifically to T, E1A, and multiple cellular proteins . Cyclin A bound to the p107 pocket, but not the RB pocket . Although both pockets contain two, related collinear subsegments (A and B), the unique sequence in the p107 pocket that occupies the space between A and B is required for the interaction with cyclin A.

Nature, 1992 Jan 2, 355(6355), 87 - 9
Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR; Ansari AZ et al.; Positive control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase . Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions . Activator-induced DNA bending is typically assigned a role in binding-site recognition, alterations in DNA loop structures or optimal positioning of the activator for interaction with polymerase . Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA . The allosterically modulated transcription factor MerR is a repressor and an Hg(II)-responsive activator of bacterial mercury-resistance genes . Escherichia coli RNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref . 6) . Chemical nuclease studies show that the activator form, but not the repressor, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site . Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33 degrees relative to the MerR-operator complex . The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.

Gene, 1992 Jan 2, 110(1), 95 - 9
The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12; Kalman M et al.; A gene is identified in the Escherichia coli K-12 spo operon as recG . Previously identified genes in the spo operon were spoS, alias rpoZ, encoding the omega (omega) subunit of RNA polymerase, as well as the spoT gene encoding the major cellular source of guanosine 3',5'-bispyrophosphate hydrolase activity . The gene order within the spo operon is: spoS (rpoZ), spoT, spoU, recG . A convergent gltS gene is present beyond the spo operon . Mutants bearing recG deletion-insertion alleles display mild sensitivities to both ultraviolet irradiation and to mitomycin C, which is expected to be due to a known recG insertion allele . Deletion-insertion mutations in upstream operon genes (spoT and spoU) show polar effects on these assays of recG function . The deduced 693-amino acid (aa) RecG sequence shows a weak, but significant, relatedness to aa sequence motifs previously reported for putative helicases involved in replication, recombination, and DNA repair.

Gene, 1992 Jan 2, 110(1), 119 - 22
Multifunctional yeast high-copy-number shuttle vectors; Christianson TW et al.; A set of four yeast shuttle vectors that incorporate sequences from the Saccharomyces cerevisiae 2 mu endogenous plasmid has been constructed . These yeast episomal plasmid (YEp)-type vectors (pRS420 series) differ only in their yeast selectable markers, HIS3, TRP1, LEU2 or URA3 . The pRS420 plasmids are based on the backbone of a multifunctional phagemid, pBluescript II SK+, and share its useful properties for growth in Escherichia coli and manipulation in vitro . The pRS420 plasmids have a copy number of about 20 per cell, equivalent to that of YEp24 . During non-selective yeast growth, pRS420 plasmids are lost through mitotic segregation at rates similar to other YEp vectors and yeast centromeric plasmid (YCp) vectors, in the range of 1.5-5% of progeny per doubling . The pRS420 series provides high-copy-number counterparts to the current pRS vectors {Sikorski and Hieter, Genetics 122 (1989) 19-27}.

Gene, 1992 Jan 2, 110(1), 101 - 3
Two cat expression vectors for cloning and generation of 3'- and 5'-deletion mutants; Kumar G; The construction of two versatile cat vectors, pGK0CAT and pGKA10CAT, is reported . These vectors possess multiple cloning sites derived from the Bluescript pKS(+) plasmid that allow the cloning of diverse DNA fragments . From a single cloned insert, i.e., a putative promoter element, one can use the exonuclease III (ExoIII) and S1 or mung-bean nuclease method to generate sequential deletion mutants of the 3' and 5' region . Linker-scanning and internal deletion mutants can thus be created by using appropriate 3'- and 5'-deletion mutants . These plasmids could thus be used for the identification of cis-acting promoter or enhancer elements by either in vivo or in vitro transcriptional analyses . The ability of these vectors to generate deletion mutants from the 3' end make them suitable to identify cis-acting elements in the 5'-noncoding region of the mRNA involved in the translational regulation of protein synthesis . Single-stranded circular mutant plasmids could also be generated from these vectors to study various protein-DNA interactions.

Gene, 1992 Jan 2, 110(1), 115 - 8
An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1; Hinshelwood S et al.; A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis . The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M . smegmatis and Mycobacterium bovis BCG . The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria . After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb . A genomic library from M . smegmatis was constructed in E . coli; clones from this library were transferred into M . smegmatis by electroporation, and back again to E . coli, without any apparent rearrangements . This vector will be useful in cloning genes encoding complex pathways in mycobacteria.

Gene, 1992 Jan 2, 110(1), 1 - 7
A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC); Kur J et al.; Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique {Koob et al., Science 241 (1988) 1084-1086} for generating rare natural cleavage sites . When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which overlapped the ihf sites) remained cleavable after prior methylation with the cognate M.EcoRI, M.HinfI, or Dam methyltransferases in the presence of IHF . Thus, IHF-AC essentially converted these enzymes into very rare cutters . The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature . Moreover, the method permits the genomic location and strength of the ihf sites to be determined.

Oncogene, 1992 Jan, 7(1), 9 - 17
c-ets-1 DNA binding to the PEA3 motif is differentially inhibited by all the mutations found in v-ets; Leprince D et al.; The proto-oncogene c-ets-1, one of the two cellular sequences transduced by the avian retrovirus E26, encodes for two transcription factors that activate through a purine-rich motif . The v-ets oncogene differs from its cellular progenitor p68c-ets-1 (i) by its fusion to gag- and myb-derived sequences in the E26 P135gag-myb-ets fusion protein, (ii) by two point mutations, and (iii) by the replacement of the 13 C-terminal amino acids present in c-ets-1 by 16 unrelated residues in v-ets . A 35 kDa protein which binds to the purine-rich PEA3 motif in a sequence-specific manner has been obtained by expression in Escherichia coli of the 311 carboxy-terminal amino acids of c-ets-1 . Using various v-/c-ets-1 chimeric 35 kDa proteins expressed in bacteria, we have shown that all the mutations found in v-ets, when introduced into this c-ets-1 protein, diminish or even abolish its sequence-specific DNA binding . These results demonstrate that, in addition to the previously defined 85 amino acids located near the carboxy terminus of the c-ets-1 protein (the ETS domain), other sequences are required for sequence-specific DNA binding . In addition, the c-ets-1 35 kDa polypeptide carrying the two point mutations and the viral-specific carboxy terminus, and thus similar to the v-ets-encoded domain of the E26 P135gag-myb-ets, does not bind to the PEA3 motif.

Life Sci, 1992, 50(11), 807 - 11
Serum phospholipase A2 enzyme activity and immunoreactivity in a prospective analysis of patients with septic shock; Vadas P et al.; Massive elevations of serum phospholipase A2 activity have been documented in patients with septic shock . Serum PLA2 activity correlated to the degree and duration of circulatory collapse, while purified native PLA2 reproduced hypotension in experimental animals . In a prospective study of patients with septic shock, we have determined the relationship of PLA2 enzyme activity to PLA2 immunoreactivity using radiolabelled E . coli phospholipid substrate and an ELISA specific for group II human nonpancreatic PLA2 . In all patients, there was a clear concordance of the two assays . Maximal PLA2 concentration was increased a mean of 554-fold over normal levels . We found no evidence to support the presence of activating or inhibitory proteins . These data confirm that the observed increase in serum PLA2 activity in septic shock is due to intravascular release of group II nonpancreatic PLA2.

EMBO J, 1992 Jan, 11(1), 71 - 7
Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli; Sherman MYu et al.; The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps) . We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation . At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK . However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis . Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES) . The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants . This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced) . In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation . The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition . Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased . Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1992 Jan, 11(1), 57 - 62
Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme; Kamitani S et al.; Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm . The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space . It was susceptible to degradation by proteases in vivo and in vitro . The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues . PhoA synthesized in the mutants indeed lacked disulfide bridges . These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.

EMBO J, 1992 Jan, 11(1), 215 - 23
Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor; Knight KL et al.; Residues 2, 6, 8 and 10 of Mnt repressor are the major determinants of operator DNA binding and recognition . Here, we investigate the interaction of wild-type Mnt and mutants bearing the Arg2----Lys, His6----Ala, Asn8----Ala and Arg10----Lys mutations with operator DNA modified by methylation or by symmetric base substitutions . The wild-type pattern of methylation interference is altered in specific ways for each of the mutant proteins . In addition, some of the mutant proteins show a 'loss of contact' phenotype with specific mutant operators . Taken together, these and previous results predict the following contacts between side chains in the Mnt tetramer and operator DNA: Arg2 recognizes the guanines at operator positions 10 and 12; His6 contacts the guanines at operator positions 5 and 17; Asn8 contacts operator positions 4, 7, 15 and 18; Arg10 contacts the guanines at operator positions 8 and 14 . The proposed contacts can be accommodated in a structural model in which the anti-parallel beta-sheet motifs of Mnt dimers lie in the major grooves of each operator half-site, centered over pseudo-symmetry axes that are 5.5 bp from the central dyad axis of the operator.

Mol Microbiol, 1992 Jan, 6(1), 115 - 21
Haemolysin-derived synthetic peptides with pore-forming and haemolytic activity; Oropeza-Wekerle RL et al.; Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990) . A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA . We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer . In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs) . The haemolytic activity of Pep1 and of E . coli haemolysin is completely inhibited by antibodies raised against Pep1.

JPEN J Parenter Enteral Nutr, 1992 Jan-Feb, 16(1), 25 - 31
Protein malnutrition alone and in combination with endotoxin impairs systemic and gut-associated immunity; Deitch EA et al.; Because protein-malnourished or endotoxemic patients are at an increased risk of developing nosocomial infections, this study was performed to investigate the effects of protein malnutrition and endotoxemia, alone and in combination, on systemic and intestinal immunity . Protein malnutrition was created by feeding the animals a solid diet containing 0.03% protein . Subgroups of these protein-malnourished mice were killed after being challenged with saline or endotoxin on days 0, 7, 14, or 21 . At death, the animals were weighed, tissues were harvested for histologic analysis (ileum, mesenteric lymph node {MLN}, liver, and spleen), mitogen responsiveness (MLN, Peyer's patches, and spleen), and xanthine oxidase measurements (ileum and cecum) . Separate groups were evaluated for survival . Both the saline and endotoxin-challenged mice had lost about 30% of their body weight after 21 days on the low-protein diet . The protein-malnourished mice were more susceptible to endotoxin-induced mortality (70% at 21 days) than the normally nourished mice (0%) (p less than .001) . The mitogen responsiveness of the protein-malnourished mice to the T-cell mitogens (PHA and Con-A) progressively decreased the longer the mice were protein malnourished, and this decreased in blastogenic responsiveness was associated with histologic evidence of lymphoid atrophy . In contrast, the blastogenic response to the primarily B-cell mitogen, PWM, was largely preserved . The endotoxin challenge further depressed the immune state of mice tested after 0, 7, or 14 (but not 21) days of protein malnutrition . Thus, both protein malnutrition and endotoxin impaired systemic and gut-associated immune responsiveness to mitogens . However, in the protein-malnourished mice, the degree of immune suppression did not correlate with endotoxin-induced mortality.

J Antimicrob Chemother, 1992 Jan, 29(1), 19 - 25
Uptake of minocycline by Escherichia coli; Chopra I et al.; Uptake of tetracyclines into Escherichia coli was assessed with a strain carrying a tetA-lacZ translational fusion, in which expression of the enzyme is controlled by the pSC101 tetR repressor gene, by examining beta-galactosidase induction . The ability of tetracycline analogues to induce beta-galactosidase synthesis was correlated with their hydrophobicity, such that hydrophobic analogues were poor enzyme inducers . Treatment of E . coli with polymyxin B nonapeptide (PMBN) rendered cells more permeable to minocycline, but not to tetracycline.

Mol Gen Genet, 1992 Jan, 231(2), 256 - 64
Regulation of the gua operon of Escherichia coli by the DnaA protein; Tesfa-Selase F et al.; The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis . Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence . Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions . In these strains beta-galactosidase acts as a reporter enzyme for transcription initiated at guaP . When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed . Reducing the intracellular level of DnaA, either by sequestration with an oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP . Thus the transcriptional activity of the gua operon is coupled, through the DnaA protein, to the DNA replication cycle . Repression of guaP by DnaA was dependent on the presence of both boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA.

Mol Gen Genet, 1992 Jan, 231(2), 248 - 55
In vitro interactions of integration host factor with the ompF promoter-regulatory region of Escherichia coli; Ramani N et al.; Previous work has shown that integration host factor (IHF) mutants have increased expression and altered osmoregulation of OmpF, a major Escherichia coli outer membrane protein . By in vitro analysis the possibility was investigated that IHF interacts directly with the ompF promoter region . Gel retardation assays and DNase I protection experiments showed that IHF binds to two sites in the ompF promoter region centered at positions -180 and -60 relative to the start of transcription . Gel electrophoresis studies with circularly permuted ompF promoter fragments indicated that IHF binding strongly increased a small intrinsic bend in the ompF promoter region . The addition of IHF to a purified in vitro transcription system strongly and specifically inhibited ompF transcription . This inhibition was reversed by increasing the concentration of OmpR, a positive activator required for ompF expression, suggesting that IHF may inhibit ompF transcription by altering how OmpR interacts with the ompF promoter.

Mol Gen Genet, 1992 Jan, 231(2), 169 - 78
Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication; Asai T et al.; Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein . In contrast, DnaA protein repressed the previously described ori-L leftward transcription . The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers . The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC . Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers . From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

Biotechniques . 1992 Jan;12(1):28, 30.
An efficient method for blunt-end ligation of PCR products; Liu ZG et al.; This report presents data demonstrating a simple method that can potentially be extended to a wide range of cloning strategies to increase the yield of insert-containing recombinants . The method requires that the ligation of an insert to a vector does not regenerate the original restriction enzyme recognition sequence . In the example presented, PCR products were blunt-end ligated to a SmaI-cut vector, in the presence of SmaI endonuclease . The addition of the restriction enzyme to the ligation reaction dramatically favored the ligation of insert to vector rather than vector self-ligation.

Scand J Immunol, 1992 Jan, 35(1), 53 - 62
Regulation of the immune response to hepatitis B virus and human serum albumin . III . Induction of anti-albumin antibody secretion in vitro by C-gene-derived proteins in peripheral B cells from chronic carriers of HBsAg; Hellstrom UB et al.; The circulatory pool of B cells from the majority (11/13) of chronic hepatitis B surface antigen (HBsAg) carriers contained sensitized B cells with the capacity to secrete IgG antibodies with specificity for human serum albumin (HSA), when stimulated with E . coli-derived core protein at low concentrations in vitro . The IgG anti-HSA secretion was dependent upon and regulated by T cells, and optimal secretion was obtained at T/B-cell ratios of 1.0-4.0, varying for different individuals . The level of anti-HSA secretion was higher for patients with on-going viral replication as assessed by hepatitis B virus (HBV)-DNA in serum . Culture supernatants containing anti-HSA antibodies also contained anti-HBc antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) where the solid phase was charged with E . coli-derived core protein, or the synthetic peptides corresponding to the 75-84 and 132-147 sequences in the C region of HBV . In contrast, IgG anti-HBc (E . coli-derived), but no anti-HSA or anti-HBc 75-84, 132-147 antibodies, were detected at similar T/B-cell ratios in cell cultures from 5/6 individuals with naturally acquired immunity to hepatitis B . These data indicate that peripheral B cells from the majority of HB-immune donors are sensitized to unique (e.g . non-albumin associated) structures in the nucleocapsid of HBV, while B cells in the majority of chronic HBsAg carriers are sensitized to linear C-gene-derived structures in association with the host 'self'-component HSA.

Hum Genet, 1992 Jan, 88(3), 320 - 4
Heterogeneity of mutations in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria; Boulechfar S et al.; Congenital erythropoietic porphyria (CEP) or Gunther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity . We have previously described two missense mutations in the UROIIIS gene, confirming that the primary defect responsible for CEP is a structural alteration of this gene . We have extended our work to 5 additional unrelated families . Two new point mutations, a deletion and an insertion have been found in the messenger RNA . Our study shows that a molecular heterogeneity of the mutations exists in Gunther's disease . One mutation (C73R), however, appears to be more frequent than the others . Finally, the different normal and mutated proteins have been expressed in Escherichia coli to determine the consequence of the mutations on the enzyme activity.

Clin Chem, 1992 Jan, 38(1), 44 - 7
New enzymatic method with tryptophanase for determining potassium in serum; Kimura S et al.; We established a simple and rapid enzymatic method for measuring potassium ion in serum by using tryptophanase (EC 4.1.99.1) purified from Escherichia coli K12 strain (E . coli K12 IFO 3301) . The presence of pyridoxal 5-phosphate promotes this enzymatic reaction, and potassium and (or) ammonium ions further accelerate it, with ammonium and potassium ions providing equivalent acceleration . We eliminated endogenous ammonium ion by using glutamate dehydrogenase (GLDH; EC 1.4.1.4), then produced ammonium ion in the presence of tryptophanase, tryptophan, and pyridoxal 5-phosphate . The concentration of formed ammonium ion, which was proportional to that of potassium ion in sample, was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then read the change of absorbance at 340 nm . The standard curve was linear for potassium ion concentrations up to 7.00 mmol/L . The within-assay variation (CV) was 0.89% at 5.51 mmol/L and 1.32% at 3.37 mmol/L . The day-to-day CVs were 0.99% at 6.85 mmol/L and 1.71% at 3.52 mmol/L . Analytical recoveries ranged from 98.7% to 108.9% . The correlation coefficient between values obtained with this enzymatic assay (y) and by flame photometry (x) was 0.995: y = 0.984x + 0.091 mmol/L (Sy.x = 0.105, n = 100) . The presence of hemoglobin, bilirubin, or other cations little affects this system.

Am J Surg, 1992 Jan, 163(1), 100 - 3; discussion 103-4
Influence of levamisole on pancreatic infection in acute pancreatitis; Widdison AL et al.; We investigated the effect of levamisole on pancreatic infection in a model of acute pancreatitis (AP) in cats . Animals with and without AP received Escherichia coli intravenously . Blood was then taken at intervals for culture . AP reduced phagocytic function by 28% as measured by the rate of bacterial disappearance from the blood (p less than 0.03) . In other cats, AP was induced, and E . coli were placed into the pancreatic duct . Levamisole was given orally in some cats; the remainder were untreated . Control cats (neither AP nor levamisole) also received E . coli . Seven days later, pancreases from all control cats were sterile . In AP cats, the pancreatic infection rate was 73% . Levamisole reduced the rate of infection to 22% (p less than 0.03) . We concluded that phagocytic function was impaired in cats with AP . Levamisole reduced the rate of pancreatic infection.

Am J Obstet Gynecol, 1992 Jan, 166(1 Pt 1), 14 - 5
Abruptio placentae associated with perforated appendicitis and generalized peritonitis; Yaron Y et al.; A primigravid woman at 35 weeks' gestation was admitted with abdominal pain, fever, and vomiting . Forceful contractions and signs of fetal distress suggested abruptio placentae . During caesarean section, seropurulent exudate and a perforated appendix were found; an appendectomy was performed . A mechanism linking appendicitis with abruptio placentae is suggested.

Genetics, 1992 Jan, 130(1), 37 - 49
Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli; Nussbaum A et al.; To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells . Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively . Plasmid recombinants were analyzed with restriction endonucleases . Our results indicate that a DSB can induce more than one type of RecE-mediated recombination . A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction . (2) Repair of the break depended on homologous sequences on the resident plasmid . (3) Break-repair was frequently associated with conversion of alleles that were cis to the break . (4) Conversion frequency decreased as the distance from the break increased . (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant . The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology . Both the cut and the uncut substrates were recipients in conversion events . Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology . We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction.

Plant Mol Biol, 1992 Jan, 18(1), 65 - 78
Activation of a truncated PR-1 promoter by endogenous enhancers in transgenic plants; Beilmann A et al.; PR-1 genes are induced by various environmental stimuli such as pathogen attack or exposure of the plants to certain chemicals . To examine the regulation of these genes, the 5' flanking regions of the PR-la gene and of two PR-1 pseudogenes were joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene . These constructs were stably integrated into the tobacco genome and independent primary transformants were monitored for the expression of the reporter gene . Unexpectedly, out of 55 transformants analysed, four plants exhibited considerable GUS activities without any inductive treatment of the plants . Expression of the endogenous PR-1 genes, however, could not be detected in these plants . Primer extension analyses revealed correct initiation of the PR1/GUS hybrid transcripts from the PR-1a TATA box . When the plants were analysed at the cellular level, clear differences regarding the tissue specificity of expression of the reporter gene were observed . These results strongly suggest that the PR1/GUS hybrid promoter expression cassettes may be activated when integrated in the vicinity of heterologous enhancer elements dispersed in the tobacco genome . In order to support this hypothesis, domain B of the enhancer of the 35S RNA promoter from cauliflower mosaic virus (CaMV) was fused to various PR1/GUS hybrid genes upstream as well as downstream from the RNA start site . These constructs were stably introduced into the tobacco genome . In any primary transformant analysed, strong GUS activities were observed with the PR1/GUS hybrid RNAs originating from the normal transcription start site of the PR-1a gene . The tissue specificity of gene expression was identical to that described previously for the CaMV 35S domain B enhancer element . Thus, modulations of the transcriptional activity of the PR-1 promoter can be achieved by heterologous enhancers in transgenic plants and may be encountered upon random integration of PR-1 promoter constructs into the tobacco genome.

Biochem J, 1992 Jan 1, 281 ( Pt 1), 57 - 65
Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity; Cook AL et al.; Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen . A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB . Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32 . Substitution of this arginine residue, or arginine-28 {a potential KEX2 (lysine-arginine endopeptidase) cleavage site}, prevents or reduces cleavage of PDGF-BB respectively . These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB . These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson {(1990) Mol . Cell Biol . 10, 5496-5501} suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding.

Biochem J, 1992 Jan 1, 281 ( Pt 1), 255 - 9
Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system . Strong synergistic effects with protein disulphide-isomerase; Soute BA et al.; It has been shown previously that the thioredoxin system (thioredoxin + thioredoxin reductase + NADPH) may replace dithiothreitol (DTT) as a cofactor for vitamin KO and K reductase in salt-washed detergent-solubilized bovine liver microsomes . Here we demonstrate that the system can be improved further by adding protein disulphide-isomerase (PDI) to the components mentioned above . Moreover, NADPH may be replaced by reduced RNAase as a hydrogen donor . In our in vitro system the various protein cofactors were required at concentrations 2-5 orders of magnitude lower than that of DDT, whereas the maximal reaction rate was about 3-fold higher . PDI stimulated the thioredoxin-driven reaction about 10-fold, with an apparent Km value of 8 microM . These data suggest that in the vitro system the formation of disulphide bonds is somehow linked to the vitamin K-dependent carboxylation of glutamate residues . In vivo, both disulphide formation and vitamin K-dependent carboxylation are post-translational modifications taking place at the luminal side of the endoplasmic reticulum of mammalian secretory cells . The possibility that the reactions are also coupled in vivo is discussed.

Arch Virol, 1992, 122(3-4), 223 - 35
Identification and sequence determination of the capsid protein gene of feline calicivirus; Carter MJ et al.; We have determined 4380 bases of the sequence from a cDNA clone containing the 3' end of feline calicivirus strain F9 . We find four candidate open reading frames of which three are complete and comprise 245, 317 and 2012 nucleotides . The fourth continues toward the 5' end . We have expressed the largest complete open reading frame in E . coli . Sera raised to this antigen react specifically with the capsid protein and its intracellular precursor molecule . N-terminal sequence analysis of purified, mature capsid protein confirms this assignment and has identified the position at which precursor is cleaved.

J Med Microbiol, 1992 Jan, 36(1), 37 - 40
A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh; Faruque SM et al.; We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea . E . coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence . With 1136 isolates from 387 patients, there was general agreement between the two assay methods . When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay . In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively . Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays . Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe . Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results . None of the other probe-positive isolates hybridised with any of the cloning vectors used . The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.

J Exp Med, 1992 Jan 1, 175(1), 275 - 84
A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein; Mehra V et al.; Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast . A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced . The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E . coli . The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M . leprae proteins tested . The degree of reactivity paralleled the response to intact M . leprae throughout the spectrum of leprosy . Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M . leprae-reactive T cell precursors responded to the 10-kD antigen . T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein . T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M . leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M . tuberculosis . Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M . leprae . The strong T cell responses against the M . leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

J Cell Biol, 1992 Jan, 116(1), 1 - 14
Localization of the nucleolar protein NO38 in amphibian oocytes; Peculis BA et al.; To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes . We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products . The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein . We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations . When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli . To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA . Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation . We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38 . This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule . Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain . Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus . We suggest that nucleolar accumulation of NO38 requires more than a single linear domain.

Genes Dev, 1992 Jan, 6(1), 129 - 34
In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions; Berzal-Herranz A et al.; In vitro selection methods provide rapid and extremely powerful tools for elucidating interactions within and between macromolecules . Here, we describe the development of an in vitro selection procedure that permits the rapid isolation and evaluation of functional hairpin ribozymes from a complex pool of sequence variants containing an extremely low frequency of catalytically proficient molecules . We have used this method to analyze the sequence requirements of two regions of the ribozyme-substrate complex: a 7-nucleotide internal loop within the ribozyme that is essential for catalytic function and substrate sequences surrounding the cleavage-ligation site . Results indicate that only 3 of the 16,384 internal loop variants examined have high cleavage and ligation activity and that the ribozyme has a strong requirement for guanosine immediately 3' to the cleavage-ligation site.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 56 - 9
parB: an auxin-regulated gene encoding glutathione S-transferase; Takahashi Y et al.; We have isolated an auxin-regulated cDNA, parB, from the early stage of cultured tobacco mesophyll protoplasts . The expression of parB was observed during transition from G0 to the S phase of tobacco mesophyll protoplasts cultured in vitro . The predicted amino acid sequence of parB cDNA has 213 amino acid residues with a relative molecular weight of 23,965 . Nucleotide sequence analysis revealed that parB cDNA has homology to glutathione S-transferase (GST; RX:glutathione R-transferase, EC 2.5.1.18) from several sources including plant and animal cells . When we introduced expression vector pKK233-2, which retains parB cDNA, into Escherichia coli, we could detect GST activity in the parB gene product . Accordingly a significant increase of GST activity was detected in the tobacco mesophyll protoplasts cultured in the presence of 2,4-dichlorophenoxyacetic acid . This is an example in which the function of auxin-regulated gene product is shown to be ascribed to a specific enzymatic activity . As GST, and its substrate glutathione, are shown to be related to cell proliferation as well as detoxification of xenobiotics in plant and animal cells, the role of parB is discussed in relation to the induction of proliferative activity in differentiated and nondividing mesophyll protoplasts of tobacco.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 397 - 401
Class II (B) general transcription factor (TFIIB) that binds to the template-committed preinitiation complex is different from general transcription factor BTF3; Moncollin V et al.; A class II (B) general transcription factor of 34 kDa has been purified from HeLa cells to apparent homogeneity . This factor appears to be transcription factor IIB (TFIIB), since it binds in vitro to template-committed preinitiation complexes formed between a template containing the TATA box/cap-site elements of the adenovirus type 2 major late promoter (Ad2MLP) and recombinant human or yeast TFIID (previously called BTF1) expressed in Escherichia coli . DNase I footprint studies show an extended pattern of protection of Ad2MLP TATA box/cap-site sequences when TFIIB is bound to template-committed complexes, even though TFIIB does not bind on its own to the template in the absence of TFIID . We also show that TFIIB is different from BTF3 by a number of criteria.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 295 - 9
Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini; Silber KR et al.; An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced . Tsp specifically degrades a variant of the N-terminal domain of lambda repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues . This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues . The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids . The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.

Mol Cell Biol, 1992 Jan, 12(1), 30 - 7
The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA; Killeen MT et al.; RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z . F . Burton, M . Killeen, M . Sopta, L . G . Ortolan, and J . F . Greenblatt, Mol . Cell . Biol . 8:1602-1613, 1988) . Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M . Sopta, Z . F . Burton, and J . Greenblatt, Nature (London) 341:410-414, 1989) . To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E . coli . Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74 . Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA . In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase . DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30 . Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

Mol Cell Biol, 1992 Jan, 12(1), 283 - 91
MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import; Atencio DP et al.; The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae . The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene . The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1 . Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures . Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures . The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C . These results indicate a role for a DnaJ cognate protein in mitochondrial protein import.

J Bacteriol, 1992 Jan, 174(2), 630 - 2
Sensitization of Escherichia coli cells to oxidative stress by deletion of the rpoH gene, which encodes the heat shock sigma factor; Kogoma T et al.; A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress . The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal . Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin . sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type.

J Bacteriol, 1992 Jan, 174(2), 558 - 67
NotI genomic cleavage map of Escherichia coli K-12 strain MG1655; Heath JD et al.; Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655 . The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al . (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E . coli DNA sequence for NotI sites . In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions . These sites were localized to clones in the lambda library and, when possible, sequenced genes . The map was compared with that of strain EMG2, a wild-type E . coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655 . Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E . coli K-12 strains.

J Bacteriol, 1992 Jan, 174(2), 456 - 63
Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein; Arini A et al.; To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication . Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay . Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites . Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids . Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent . A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity . A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population . A mutation in the middle RS2 site had no significant effect on pSC101 replication.

J Bacteriol, 1992 Jan, 174(2), 415 - 25
cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth; Neuwald AF et al.; The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite . Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes . cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene . A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs . The auxotrophy caused by cysQ null mutations was leaky in some but not all E . coli strains and could be compensated by mutations in unlinked genes . cysQ mutants were prototrophic during anaerobic growth . Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis . Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects . cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport . Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E . coli and the gene for mammalian inositol monophosphatase . Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.

J Bacteriol, 1992 Jan, 174(1), 63 - 70
Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli; de Boer PA et al.; The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins . In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division . MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles . At normal levels of expression, both MinC and MinD are required for the division block . We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD . The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor . The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor.

J Bacteriol, 1992 Jan, 174(1), 35 - 9
New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways; Mulder E et al.; Proper positioning of division sites in Escherichia coli requires balanced expression of minC, minD, and minE gene products . Previous genetic analysis has shown that either MinD or an apparently unrelated protein, DicB, cooperates with MinC to inhibit division . We have isolated and sequenced minC mutations that suppress division inhibition caused by overproduction of either DicB or MinD proteins . Most missense mutations were located in the amino acid 160 to 200 region of MinC (231 amino acids) . Some mutations exhibited preferential resistance to one or the other coinhibitor, suggesting that two distinct proteins, possibly MinD and DicB themselves, interact in slightly different manners with the same region of MinC to promote division inhibition.

J Bacteriol, 1992 Jan, 174(1), 320 - 3
In vivo inhibition of TonB-dependent processes by a TonB box consensus pentapeptide; Tuckman M et al.; The TonB box, a conserved pentapeptide sequence found in TonB-dependent colicins and receptors, is thought to interact physically with the TonB protein to facilitate TonB-dependent processes . Strains of Escherichia coli were treated in vivo with the synthetic TonB box pentapeptide Glu-Thr-Val-Ile-Val . The pentapeptide inhibited several TonB-dependent processes, including cell growth in low-iron medium, phi 80 infection, and killing by colicins B and Ia . Two unrelated control pentapeptides had no effect on TonB-dependent processes.

J Bacteriol, 1992 Jan, 174(1), 315 - 9
Sequence and expression of the Escherichia coli K1 neuC gene product; Zapata G et al.; The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids . A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified . Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair . Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.

J Bacteriol, 1992 Jan, 174(1), 233 - 40
Integration host factor is required for positive regulation of the tdc operon of Escherichia coli; Wu YF et al.; A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E . coli integration host factor (IHF) . In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain . Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level . Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF . In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter . IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence . These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.

J Bacteriol, 1992 Jan, 174(1), 122 - 9
Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti; Matsushita K et al.; Acetobacter aceti has an ability to grow under two different culture conditions, on shaking submerged cultures and on static pellicle-forming cultures . The respiratory chains of A . aceti grown on shaking and static cultures were compared, especially with respect to the terminal oxidase . Little difference was detected in several oxidase activities and in cytochrome b and c contents between the respiratory chains of both types of cells . Furthermore, the results obtained here suggested that the respiratory chains consist of primary dehydrogenases, ubiquinone, and terminal ubiquinol oxidase, regardless of the culture conditions . There was a remarkable difference, however, in the terminal oxidase, which is cytochrome a1 in cells in shaking culture but cytochrome o in cells grown statically . Change of the culture condition from shaking to static caused a change in the terminal oxidase from cytochrome a1 to cytochrome o, which is concomitant with an increase of pellicle on the surface of the static culture . In contrast, reappearance of cytochrome a1 in A . aceti was attained only after serial successive shaking cultures of an original static culture; cytochrome a1 predominated after the culture was repeated five times . In the culture of A . aceti, two different types of cells were observed; one forms a rough-surfaced colony, and the other forms a smooth-surfaced colony . Cells of the former type predominated in the static culture, while the cells of the latter type predominated in the shaking culture . Thus, data suggest that a change of the culture conditions, from static to shaking or vice versa, results in a change of the cell type, which may be related to the change in the terminal oxidase from cytochrome a1 to cytochrome o in A . aceti.

Infect Immun, 1992 Jan, 60(1), 63 - 70
Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis; Connell TD et al.; Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite . Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes . Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity . Mutant alleles that determined altered binding specificities were sequenced . Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa . Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1 . Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34 . Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.

Infect Immun, 1992 Jan, 60(1), 13 - 8
Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (O126:H27); Benz I et al.; The adherence of diarrhea-associated Escherichia coli to the small-bowel mucosa is an important step in the pathogenesis of diarrheal diseases . In tissue culture systems, diarrhea-associated strains show three distinct patterns of adherence: localized adherence, diffuse adherence (DA), and the recently described aggregative adherence . To study the molecular basis of the DA phenotype, we investigated the diarrhea-associated DA strain 2787 (O126:H27), isolated from a case of infantile diarrhea . The DA phenotype is mediated by a 6.0-kb DNA fragment derived from a 100-kb plasmid harbored by the wild-type strain . This fragment codes for a 100-kDa protein which can be released from the bacterial cell into the supernatant by mild heat treatment . Recombinant DA+ strains as well as the isolated 100-kDa protein were used to engender specific antisera in rabbits . As demonstrated by Western blotting (immunoblotting), the antibodies engendered by the recombinant DA+ strain recognized a 100-kDa protein in the wild-type strain 2787 and in all recombinant strains showing DA . Immunogold electron microscopy localized the 100-kDa protein to the bacterial cell surface . Serologically related proteins of similar size were detected by Western blotting in other DA+ diarrhea-associated strains belonging to enteropathogenic E . coli serotypes . The 100-kDa protein denoted AIDA-I (adhesin involved in diffuse adherence) binds in a saturable fashion to HeLa cells . AIDA-I-specific immunoglobulin G antibodies--and, to an even greater extent, Fab fragments derived thereof--inhibited bacterial attachment to HeLa cells . This is direct evidence that the 100-kDa protein is the adhesin mediating the DA phenotype of these diarrhea-associated strains and is representative of a group of serologically related proteins in other DA+ strains.

Crit Care Med, 1992 Jan, 20(1), 119 - 25
Longitudinal distribution of pulmonary vascular resistance after endotoxin administration in sheep; Pearl RG et al.; BACKGROUND AND METHODS: Pulmonary hypertension may increase pulmonary capillary pressure and exacerbate pulmonary edema in acute respiratory failure . The effects of pulmonary hypertension on pulmonary capillary pressure depend on the longitudinal distribution of pulmonary vascular resistance . Since pulmonary hypertension occurs during acute respiratory failure, we hypothesized that acute respiratory failure may produce time-dependent changes in the longitudinal distribution of pulmonary vascular resistance . Therefore, we measured pulmonary capillary pressure and the longitudinal distribution of pulmonary vascular resistance in an animal model of acute respiratory failure . Escherichia coli endotoxin (2.5 to 5.0 micrograms/kg) was administered over a 1-hr period in eight anesthetized sheep . Pulmonary and systemic hemodynamics, including pulmonary artery occlusion pressure (PAOP), pulmonary capillary pressure, and the longitudinal distribution of pulmonary vascular resistance, were measured over the next 5 hrs . Pulmonary capillary pressure was estimated by analysis of the pressure decay following pulmonary artery balloon inflation . RESULTS: Endotoxin administration resulted in sustained pulmonary hypertension for the subsequent 5 hrs of the study . Pulmonary capillary pressure was increased 7 mm Hg above baseline at 0.5 and 0.75 hrs during the infusion of endotoxin but returned to baseline values at 1.5 hrs . Despite sustained pulmonary hypertension, pulmonary capillary pressure remained at baseline values for the duration of the study . Similar to pulmonary capillary pressure, pulmonary venous (or postcapillary) resistance was increased approximately four-fold over baseline at 0.5 and 0.75 hrs after initiating endotoxin administration, but returned to baseline values by the end of endotoxin administration and remained at baseline values throughout the remainder of the study . In contrast, pulmonary arterial (or precapillary) resistance remained at values approximately three times baseline during the infusion and throughout the duration of the study . CONCLUSIONS: In this experimental model of acute respiratory failure, the effects of endotoxin on the longitudinal distribution of pulmonary vascular resistance are time-dependent . If these data from animals can be extrapolated to humans, we speculate that the importance of pulmonary venoconstriction in exacerbating pulmonary edema may vary over time in patients with acute respiratory failure.

Dev Biol, 1992 Jan, 149(1), 8 - 15
Purification and characterization of maturation-promoting factor in fish; Yamashita M et al.; Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes . We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12 . The final preparation was purified over 1000-fold with a recovery of about 1% . On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa . SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa . A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates . The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody . When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein . The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not . These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.

Dig Dis Sci, 1992 Jan, 37(1), 47 - 52
Effect of base precursors on water and electrolyte transport during oral hydration solution perfusion in secreting rat intestine; Rolston DD et al.; In situ steady-state, single-pass small intestine perfusions in rats were carried out to compare the effect of the bicarbonate and citrate World Health Organization oral rehydration solutions and a base precursor-free solution on intestinal water and electrolyte transport after inducing intestinal secretion with purified heat-stable Escherichia coli enterotoxin . When toxin was not perfused, the rates of water, sodium, and bicarbonate absorption were significantly greater from the bicarbonate-containing solution than from the citrate or base precursor-free solutions . Chloride absorption was greater from the base precursor-free solution, but this might reflect the higher chloride concentration of the perfusate . When toxin was perfused, there was no significant difference among the solutions in the rates of water, potassium, or chloride absorption . Sodium absorption occurred at significantly greater rates from both the bicarbonate and the base precursor-free solutions than from the citrate solution . Base precursor-containing solutions may not provide any advantage over a base precursor-free solution in stimulating water and sodium absorption in 5'-cyclic guanosine monophosphate mediated acute diarrhea.

Surgery, 1992 Jan, 111(1), 79 - 85
Effect of dobutamine infusion on endotoxin-induced lipid peroxidation in awake sheep; Demling RH et al.; beta-agonists are known to not only increase oxygen delivery, but also attenuate the inflammatory response . We studied the effect of infusing the beta-agonist, dobutamine, on the oxidant-induced lung and liver tissue lipid peroxidation seen after endotoxemia . Twelve unanesthetized adult sheep with lung and soft tissue (prefemoral) lymph fistulae were given 5 micrograms/kg of Escherichia coli endotoxin intravenously . In six sheep, dobutamine 10 to 15 micrograms/kg/min was infused beginning 3 hours after endotoxin to increase oxygen delivery by 75% above baseline . Animals were killed at 6 hours, and lung and liver lipid peroxidation, measured as malondialdehyde, was obtained . Data were compared to six control sheep . Endotoxin alone produced increased lung and soft tissue vascular permeability as evidenced by a twofold increase in protein-rich lymph flow . Lung and liver malondialdehyde increased to 116 +/- 40 nmol/gm and 202 +/- 64 nmol/gm, respectively, compared to control values of 42 +/- 7 nmol/gm and 110 +/- 20 nmol/gm, respectively . Dobutamine infusion after endotoxin increased oxygen delivery by 75%, although changes in total oxygen consumption were not different from those seen with endotoxin alone . Lung and soft tissue lymph flow did not change with dobutamine . However, lung malondialdehyde was 41 +/- 17 nmol/gm, not different from controls . Liver malondialdehyde remained elevated at 164 +/- 26 nmol/gm . We conclude that dobutamine infusion prevents further oxidant-induced lung tissue lipid peroxidation but does not reverse the increased permeability already present . Liver lipid peroxidation was not decreased, suggesting the liver oxidant process may not be caused by the same mechanism as the lung lipid peroxidation.

J Infect Dis, 1992 Jan, 165(1), 141 - 3
Tissue culture-adherent Escherichia coli in infantile diarrhea; Echeverria P et al.; To determine the association of tissue culture-adherent Escherichia coli with diarrhea, serotyped E . coli strains isolated in a yearlong case-control study of infantile diarrhea in Bangkok, Thailand, were examined for adherence to HeLa cells and for hybridization with the enteropathogenic E . coli adherence factor, the F1845, and the enteroaggregative E . coli (EAggEC) DNA probes . E . coli that adhered to HeLa cells in a localized adherence (LA) pattern (LA E . coli) was isolated from 26 of 509 infants with diarrhea (cases) and 9 of 509 age-matched controls (P = .006); E . coli with diffuse or aggregative adherence (DA or AA) to HeLa cells or that hybridized with the F1845 or EAggEC probes was not associated with infantile diarrhea . LA E . coli of classical enteropathogenic E . coli (EPEC) serotypes was isolated from 11 cases and 1 control (P = .003) . EPEC O44:H18 that adhered to HeLa cells in a DA pattern and hybridized with the F1845 DNA probe was the predominant E . coli (five of five colonies tested) isolated from a 5-month-old girl with diarrhea in whom no other enteric infections were identified . Although LA E . coli was highly associated with infantile diarrhea, the role of DA and AA E . coli was uncertain in this setting.

Hepatology, 1992 Jan, 15(1), 122 - 9
The acute-phase response protects mice from D-galactosamine sensitization to endotoxin and tumor necrosis factor-alpha; Alcorn JM et al.; D-Galactosamine is an hepatocyte-specific inhibitor of RNA synthesis . It has been used to sensitize animals both to the lethal effects of bacterial endotoxin (lipopolysaccharide) and to a principal lipopolysaccharide-induced mediator of shock, tumor necrosis factor-alpha . The mechanism by which this sensitization occurs is unknown . Because lipopolysaccharide, acting through a network of cytokines, provokes the transcription of a number of hepatic acute-phase proteins, we postulated that the lipopolysaccharide-sensitizing effect of D-galactosamine could be caused by its inhibition of acute-phase product transcription . We confirmed that the acute-phase response to lipopolysaccharide was attenuated by simultaneous administration of D-galactosamine . However, when the acute-phase response was induced by subcutaneous turpentine 24 hr before D-galactosamine administration, the effect of D-galactosamine on circulating acute-phase reactants was negligible . Furthermore, induction of an a priori acute-phase response protected mice from both D-galactosamine/lipopolysaccharide and D-galactosamine/tumor necrosis factor-alpha-induced death . The turpentine-induced acute-phase response did not decrease endogenous tumor necrosis factor-alpha production after lipopolysaccharide, nor did it affect the clearance of larger doses of injected tumor necrosis factor-alpha . Thus we suggest that the acute-phase response protects against death in D-galactosamine-sensitized mice through an interaction with mediators of shock subsequent to tumor necrosis factor-alpha release.

Diabetes, 1992 Jan, 41(1), 118 - 21
T-lymphocyte lines specific for glutamic acid decarboxylase (GAD) the 64K beta-cell antigen of IDDM; Diaz JL et al.; Insulin-dependent diabetes mellitus (IDDM) is viewed as a thymus-dependent autoimmune disease, although the specific beta-cell autoantigen or autoantigens remain unknown . The recent identification of the beta-cell 64K antigen as the enzyme glutamic acid decarboxylase (GAD) permits investigation of GAD as a candidate for the autoantigen associated with beta-cell destruction, mediated by T-lymphocytes, in susceptible individuals . In this study, we describe the isolation of GAD-specific T-lymphocyte lines from BB rats, an animal model of IDDM . GAD (Escherichia coli) was inoculated into the footpads of diabetes-resistant BB rats, and after 10 days, a popliteal lymph node cell culture suspension was prepared . GAD-specific T lymphocytes were obtained by culture with interleukin 2 and repeated stimulation with GAD in the presence of BB rat thymic antigen-presenting cells . Four stable, CD4+, MHC (RT1u)-restricted T-lymphocyte lines were isolated . They proliferate selectively in the presence of GAD and secrete interleukin 2 and interferon-gamma . T-lymphocyte lines such as these could be important in the definition of pathogenetic epitopes associated with GAD.

Arch Biochem Biophys, 1992 Jan, 292(1), 34 - 41
Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits; Lim WK et al.; Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein . Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins . The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit . Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation . Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites . DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies . Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C . Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C) . In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C) . Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible . The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.

Arch Biochem Biophys, 1992 Jan, 292(1), 156 - 64
Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction; Van Langendonckt A et al.; A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea . In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa) . A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not . In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP-thioredoxin reductase (NTR) were evidenced . Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs . Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs . Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak . The algal thioredoxin h was also slightly active with spinach and E . coli NTRs . These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments.

Virology, 1992 Jan, 186(1), 300 - 2
Localization of a surface domain of the capsid protein of barley yellow dwarf virus; Rizzo TM et al.; We describe a method for localizing protein domains situated at the surface of virus particles . A cDNA clone of the New York PAV isolate (NY-PAV) of barley yellow dwarf virus (BYDV) containing the capsid protein gene was generated and sequenced . A defined set of overlapping cDNA fragments specific to the capsid protein ORF of NY-PAV was subcloned into the pGEX expression vectors . Cells of Escherichia coli carrying these plasmids synthesize recombinant glutathione S-transferase/capsid proteins . These proteins were used in immunoblot experiments to localize the epitopes of three PAV-BYDV-directed monoclonal antibodies to a 20 amino acid-long segment of the NY-PAV capsid protein . All three monoclonal antibodies reacted with NY-PAV virions in a triple antibody sandwich enzyme-linked immunosorbent assay, indicating that their epitopes are located at the virion surface.

J Virol, 1992 Jan, 66(1), 386 - 98
Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans; Demkowicz WE et al.; Vaccinia virus (VV) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known . By screening a lambda gt11 expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa . It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope . All of these proteins were synthesized at late times postinfection . Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively . The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697 . DNA sequence analysis, in comparison with the known nucleotide sequence of VV, provided identification of the corresponding open reading frames . Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains . Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics . The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively . Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge . Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice . Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins.

Exp Cell Res, 1992 Jan, 198(1), 107 - 14
Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells; Kido M et al.; We tried to make a well-characterized bacterial protein function in mammalian cell nuclei . For this purpose we chose Escherichia coli RecA protein and fused its carboxy terminus to the nuclear location signal of SV40 large T-antigen by oligonucleotide-dependent modification of the gene . When injected into the cytoplasm, the modified RecA protein (T-RecA for the T-antigen signal) accumulated efficiently in the nuclei, whereas the wild-type RecA protein remained in the cytoplasm . The T-RecA protein retained its original in vivo activity, judging from the finding that uv-sensitive bacteria (recA- E . coli) became uv-resistant on transformation with the T-recA plasmid as well as the recA plasmid . For expression of the T-recA gene in mammalian cells, the 5' region was replaced by the chicken beta-actin promoter and Kozak's initiation signal . A high level of expression was observed when Chinese hamster ovary (CHO-K1) cells were transfected with this plasmid . Indirect immunofluorescence examination revealed that the T-RecA protein in nuclei of mammalian cells bound to chromatin.

Arch Virol, 1992, 125(1-4), 15 - 23
Transient expression of the coat protein of sugarcane mosaic virus in sugarcane protoplasts and expression in Escherichia coli; Smith GR et al.; The coat protein (CP) of strain SC of sugarcane mosaic virus (SCMV-SC) was expressed transiently in sugarcane protoplasts after electroporation with one of two plasmids encoding the CP gene . The CP gene was fused with either the cauliflower mosaic virus 35S promoter or the synthetic monocotyledon promoter "Emu" . The coat protein gene was also inducibly expressed in Escherichia coli when fused to the trc promoter . The protein expressed in both systems had the same electrophoretic mobility and antigenic specificity as purified SCMV-SC coat protein . Transient expression of the 35S-CP gene in protoplasts could only be demonstrated in Western blots developed with the chemiluminescence enzyme substrate luminol.

Res Microbiol, 1992 Jan, 143(1), 55 - 65
Molecular typing of Brucella with cloned DNA probes; Grimont F et al.; Brucella constitutes a single genomic species (B . melitensis); however, for epidemiological studies, methods are needed for discriminating strains within this genomic species . DNA samples from 112 Brucella strains were cleaved by restriction endonucleases and the fragments separated by agarose gel electrophoresis and transferred to nylon membranes . When the DNA fragments on the membranes were probed with 32P-labelled 16 + 23 S rRNA from Escherichia coli, a single rRNA gene restriction pattern was obtained after cleavage with all endonucleases tested (HindIII, EcoRI, SmaI, and XhoI) except BamHI . This indicated high genomic homogeneity within the single Brucella species . Of 30 probes consisting of random Brucella DNA fragments cloned into lambda EMBL3, 20 yielded a single BamHI restriction pattern per probe when applied to 112 Brucella DNA tested . However, 7 probes yielded 3 to 12 different patterns among DNA tested . These patterns more-or-less correlated with the classification of strains into biogroups (Melitensis, Abortus, Suis, Neotomae, Ovis and Canis) and biovars (18 biovars represented) . Probe A was capable of separating biogroup Melitensis from the other biogroups . Probe C separated the set of biogroups Melitensis-Abortus-Ovis from the other biogroups . By reference to the patterns obtained using 1 to 7 probes, the most frequently occurring biovars (Melitensis 1, Melitensis 3, Abortus 1, Abortus 3, Suis 2 and Ovis) could be distinguished from each other . Eight biovars showed more than one pattern with 1 to 7 probes . The proposed typing system should be useful for epidemiological subtyping and does not pose safety problems once the DNA has been extracted.

Res Microbiol, 1992 Jan, 143(1), 113 - 6
Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system; Jacobson GR; The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates . An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coli mannitol permease, appears to be necessary for this protein to optimally carry out these functions . Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannitol in E . coli involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer . Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer . The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally.

Neuroscience, 1992, 47(2), 481 - 5
Interleukin-1 alpha expression is inducible by cholinergic stimulation in the rat adrenal gland; Andersson C et al.; Interleukin-1-like immunoreactivity has earlier been demonstrated by immunohistochemistry in the noradrenaline-containing chromaffin cells of the rat adrenal gland {Schultzberg et al . (1989) Neuroscience 30, 805-810; Schultzberg et al . (1987) J . Neurosci . Res . 18, 184-189} . In this study, we examine the regulation, upon cholinergic stimulation, of the expression of the cytokine interleukin-1 alpha in the rat adrenal gland . Interleukin-1 alpha and interleukin-1 alpha mRNA levels in the adrenal gland are affected by systemic administration of the cholinergic agonists nicotine (0.5 mg/kg, i.p.) and carbachol (0.5 mg/kg, i.p.) . Both drugs cause an increase in interleukin-1 alpha mRNA levels . In contrast to the increased mRNA levels, nicotine and carbachol reduce the interleukin-1 alpha protein level measured in the rat adrenal gland: nicotine by approximately 30%, 60 min after injection, and carbachol by approximately 55%, 30 min after injection . The interleukin-1 alpha protein level returns to control level 90 min after nicotine injection, and 120 min after carbachol injection . We thus found a large, constitutively expressed and inducible pool of interleukin-1 alpha in the rat adrenal gland, which appears to be sensitive to cholinergic stimulation and which may be responsible for some of the local and systemic effects of interleukin-1 alpha . Experiments with Escherichia coli lipopolysaccharide show that this substance, which induces interleukin-1 expression and secretion in macrophages, is also able to induce the expression of interleukin-1 alpha mRNA and interleukin-1 alpha in the adrenal gland when injected at the dose of 2 mg/kg, i.p.

Int J Pept Protein Res, 1992 Jan, 39(1), 77 - 81
High level synthesis of biologically active recombinant trichosanthin in Escherichia coli; Zhu RH et al.; Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase-directed promoter . Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions . Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein . Its IC50 was approximately 0.13 nM in an in vitro rabbit reticulocyte translational system and a dose of around 35 micrograms protein per 25 g body weight was sufficient to induce complete abortion in mice . Form II rTCS had a propeptide of 19 aa at the C-terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.

Cytometry, 1992, 13(5), 540 - 4
Aggregation of Escherichia coli B/r A during agar filtration: effect on morphometric measurements; Vardi E et al.; We have investigated the phenomenon of particle aggregation in a sample of 71,038 Escherichia coli B/r A cells in balanced exponential growth, during preparation for electron microscopy by agar filtration . The bacteria were photographed in a transmission electron microscope and the dimensions and spatial relationships among all the members of each aggregate were recorded using an interactive image processing system . The proportion of aggregated cells, 22%, is much greater than that found by direct count in a light microscope (7%), implying that most aggregation takes place during the preparation stages . The aggregated cells are about 1% narrower than the free cells, because of mutual compression, and 1.5% longer, because of a selection bias in favor of longer cells . From a statistical analysis of the data, we conclude that the clustering of cells into aggregates in the course of sample preparation is the result of random encounters during the settling on the collodion membrane and of the changing surface tension during the drying process . A method is proposed to correct morphometric measurements for the distortion caused by cellular aggregation of this kind.

Proc Natl Sci Counc Repub China B, 1992 Jan, 16(1), 1 - 5
Endonuclease A degrades chromosomal and plasmid DNA of Escherichia coli present in most preparations of single stranded DNA from phagemids; Lin JJ; With E . coli, large and variable amounts of chromosomal and plasmid DNAs are observed in the supernatants of overnight cultures when the cells carry an endA mutation, but are not detected by gel electrophoresis when the cells carry the wild type allele of endA . Significant amounts of nuclease activity in DH11S endA+ supernatants were detected by two simple assays; the rapid degradation of added pBR322 plasmid DNA, as judged by agarose gel electrophoresis, and a decrease of more than 100000 fold in transformation efficiency of the added pBR322 plasmid DNA . By employing isogenic endA mutant and wild type strains of DH11S and DH10B/F' proAB+ laclq Z delta M15, it was shown that detectable levels of chromosomal and plasmid DNAs are observed only in the endA mutant strains . These results indicate that Endonuclease I activity is responsible for degradation of chromosomal and plasmid DNA usually present in preparations of ssDNA . Therefore, a wild type endA gene is useful for the rapid and simple production of highly purified ssDNA from cells containing phagemid vectors.

Protein Eng, 1992 Jan, 5(1), 101 - 4
Studies on chimeric fusion proteins of human aldolase isozymes A and B; Takasaki Y et al.; Several kinds of fusion proteins between human aldolases A and B were prepared by recombinant DNA technology and their enzymic properties were examined . AB chimeras, which have aldolase A at the N-terminal region and aldolase B at the C-terminal region, were scarcely obtained, while BA chimeras were abundant (Kitajima et al., (1990), J . Biol . Chem., 265, 17493-17498) . All the BAB chimeras, aldolase A fragments inserted in aldolase B, showed activity assignable to aldolase B type, which imply an essential role of Tyr residue at the C-terminus of aldolase A in the binding of fructose-1,6-bisphosphate (Fru-1,6-P2) . BAB chimeras also showed reactivity to effectors such as fructose-2,6-bisphosphate (Fru-2,6-P2) and pyridoxal 5-phosphate (PLP), in a similar manner to aldolase B . BAB108 has a similarity to the BA108 chimera, but acts differently from other BAB chimeras, suggesting that its structure around active site looks like that of aldolase A.

Scand J Urol Nephrol, 1992, 26(2), 197 - 9
Spontaneous bladder rupture after colocystoplasty . Case report; Mansi MK et al.; An 18-year-old girl had bladder extrophy managed by sigmoid cystoplasty with clean intermittent catheterization . Spontaneous bladder rupture occurred 12 months after reconstructive surgery . The diagnosis was made by ultrasound with abdominal tapping and cystography under fluoroscopy . Management included intravenous antibiotics, laparotomy and closure of the perforation . The diagnosis was delayed and postoperative intraperitoneal abscess formation occurred.

Int J Food Microbiol, 1992 Jan-Feb, 15(1-2), 131 - 43
Evaluation of a commercial process for collection and cooling of beef offals by a temperature function integration technique; Gill CO et al.; The hygienic adequacy of a commercial process for the collection and cooling of beef offals was assessed by a temperature function integration technique . The diverse operations for collection of offals were inspected . The rates of product movement through those operations, and the temperatures of products at the time of their being packed, were determined . From that information, four of the nine product types were selected for examination of their temperature histories during the assembly and cooling of the cartoned products . The products selected encompassed product at near-body and near-air-ambient temperatures at the time of packaging, product in the largest and smallest cartons used in the process, and product with relatively short and long residence times in an unchilled carton stack assembly area . Twenty-one temperature histories were collected for each of the products, and the possible proliferation of an indicator organism, Escherichia coli, calculated for each temperature history . The results were assessed against a temperature function integration criterion derived from studies of beef carcass and cartoned meat cooling processes . Products packaged at near-ambient temperature readily met with the criterion, but products packed at near-body temperatures did not comply . The latter non-compliance was extreme for product packaged in large cartons . However, the principal cause of non-compliance was identified as highly variable cooling conditions in the carton freezing facility . A brief survey of air speeds and temperatures within that facility indicated means by which product cooling could be better controlled.

Vaccine, 1992, 10(8), 512 - 8
Immunization of pigs against Actinobacillus pleuropneumoniae with two recombinant protein preparations; Rossi-Campos A et al.; Two Actinobacillus pleuropneumoniae serotype 7 antigens were expressed in Escherichia coli and tested for their protective efficacy in an experimental pig model . The antigens used were a fusion protein containing the carboxy-terminal 70% of the +/- 103 kDa cytolysin and a full length 60 kDa protein which has been shown previously to bind transferrin . Pigs were immunized twice with 25 micrograms of either or both preparations . All pigs developed a strong humoral immune response comparable to that induced by infection . Upon challenge with an A . pleuropneumoniae serotype 7 strain, all immunized groups were less affected by the disease and showed significantly lower mortality than the controls (p less than 0.1) . Pigs receiving both antigens had a tendency to recover faster than the controls or animals which were vaccinated with only one antigen . Protection was serotype-specific since no cross-protection was detected following challenge with an A . pleuropneumoniae serotype 1 strain . A dose-response experiment using the single antigens at 200, 50 or 12.5 micrograms per dose showed no difference in protection among the groups.

J Tongji Med Univ, 1992, 12(1), 11 - 6
Effect of endotoxin on hypoxic pulmonary vasoconstriction--the role of prostaglandins and leukotrienes; Fan M et al.; In this study, we observed the effect of endotoxemia on hypoxic pulmonary vasoconstriction (HPV) in dogs and explored roles played by prostaglandins and leukotrienes in this process . 5 micrograms/kg BW of E . coli endotoxin induced transient rise in pulmonary arterial pressure and pulmonary vascular resistance (PVR) . 30 min after injection of endotoxin when PVR tended to decline, pulmonary vasoconstriction response to alveolar hypoxia was lost, and the ratio of TXB2 to 6-keto-PGF1 alpha decreased significantly . HPV was enhanced at 60-100 min and then returned to the control level at 2 h after injection of endotoxin . At these periods the ratio of TXB2 to 6-keto-PGF1 alpha was the same as before use of endotoxin, whereas plasma concentration of leukotrienes was markedly increased . Indomethacin could prevent the early loss of HPV, but no effect on the late increment of HPV was found . Diethylcarbamazine, which blocked the production of leukotrienes after use of endotoxin, could inhibit late increment of HPV . We concluded that the early loss of HPV was related to the vasodilator prostacyclin, and the late increment of HPV was mainly brought about by leukotrienes.

Pediatriia, 1992, (1), 33 - 7
{Problem of infection in fetuses and newborn infants}; Nazarov VG et al.; Overall 76 histories of births, case reports of neonates and protocols of autopsies of the dead because of infection were analyzed . It has been established that the overwhelming majority of the children had contracted infection antenatally or during birth, with the ascending pathway of infection being predominant . Attention is drawn to the clinical and postnatal diagnosis with the aid of the screening tests.

J Biochem (Tokyo), 1992 Jan, 111(1), 87 - 90
Alteration of the carbohydrate-binding specificity of the Bauhinia purpurea lectin through the preparation of a chimeric lectin; Yamamoto K et al.; A chimeric lectin gene was constructed by using a cDNA clone coding the Bauhinia purpurea lectin (BPA) in which a part of the metal-binding region was replaced by the corresponding region of the mannose-binding Lens culinaris lectin (LCA) . The chimeric lectin expressed in Escherichia coli was found to bind alpha mannosyl-bovine serum albumin (BSA) and this binding was inhibited by mannose.

J Biochem (Tokyo), 1992 Jan, 111(1), 37 - 45
Dihydrofolate reductase as a new "affinity handle"; Iwakura M et al.; Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins . The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity . Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives . All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity . The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step . The use of methotrexate-bound affinity chromatography was very effective . In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence . The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system . Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.

J Biochem (Tokyo), 1992 Jan, 111(1), 31 - 6
Dihydrofolate reductase gene as a versatile expression marker; Iwakura M et al.; The Escherichia coli dihydrofolate reductase (DHFR) gene has been used as a genetic marker specifying trimethoprim resistance (TmpR) . In order to use the DHFR gene as a versatile expression marker, we have constructed three types of plasmids: promoter cloning vector, terminator cloning vector, and the plasmid containing the DHFR gene cassette . In these systems, the selection of recombinant plasmids was carried out just by examining the TmpR phenotype of the transformed cells . Then, levels of the enzymatic activity of DHFR were measured to evaluate the efficiency of promoters and terminators in the fused DNA fragment . An expression plasmid which resulted in the E . coli host cells being able to produce DHFR up to 20% of total cellular proteins was also constructed by changing the promoter and Shine-Dalgarno sequences of the DHFR gene.

J Biochem (Tokyo), 1992 Jan, 111(1), 123 - 8
Human long-chain acyl-CoA synthetase: structure and chromosomal location; Abe T et al.; A complementary DNA clone encoding the entire human long-chain acyl-CoA synthetase was isolated and the total 698-amino acid sequence was deduced . The amino acid sequence of human long-chain acyl-CoA synthetase shows 84.9% identity to that of rat long-chain acyl-CoA synthetase . The nucleotide sequences of the protein coding regions between human and rat long-chain acyl-CoA synthetase mRNAs are highly conserved (85.6%), whereas those of the 3' untranslated regions are less conserved (72%) . The location of the human long-chain acyl-CoA synthetase gene was identified on chromosome 4 by spot hybridization of flow-sorted chromosomes . Computer-assisted homology search revealed a significant similarity of the enzyme with the enzymes of the luciferase family . Based on this similarity, the structure of human long-chain acyl-CoA synthetase can be divided into five domains: the N-terminus, two domains similar to those in enzymes of the luciferase family, a long gap region between the similar domains and the C-terminus.

Environ Mol Mutagen, 1992, 19(4), 288 - 96
Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12; Abril N et al.; In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents . The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies . By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs . methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase) . Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein . These results support the reported in vitro substrate specificities for both ogt and ada ATases . The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase . Deficient bacterial derivatives showed, by contrast, linear mutation induction responses . The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10) . The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives . This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation . Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected . In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Acta Chem Scand, 1992 Jan, 46(1), 97 - 9
Specific carbon-13 labelling of leucine residues in human growth hormone; Christensen T et al.; Biosynthetic human growth hormone specifically 13C-labelled in the carbonyl positions of all 26 leucine residues has been obtained by recombinant DNA techniques using 13C-labelled leucine and an E . coli strain that requires leucine . It is shown that, on the whole, the labelling is specific with no significant mislabelling as would have been the case had the 13C-labelled leucine been metabolized.

Yi Chuan Xue Bao, 1992, 19(1), 86 - 92
{Multi-origin usage for chromosome replication of suppressive integration strain of dnaA46 mutant of Escherichia coli integrated with R6K}; Lu J et al.; The chromosome of temperature sensitive initiation mutant dnaA46 of Escherichia coli K-12 fails to replicate at 42 degrees C . Suppressive integration (Sin) strain integrated with the R6K plasmid was screened at 42 degrees C . Marker frequency determination of the Sin strain reveals that replication was initiated at the normal site of initiation at 30 degrees C, while at three different sites at 42 degrees C . Two of the sites have been reported in the stable DNA replication mutant, one of them is a novel site . Inhibition of chromosome replication was not observed for the recA derivative of Sin initiated at two of the initiation sites close to the normal initiation site . Inhibition was observed for chromosome replication initiated near the site where chromosome replication normally terminates . It indicates that chromosome replication initiated at sites close to the terminus is recA gene dependent.

Yi Chuan Xue Bao, 1992, 19(1), 76 - 85
{Genetic recombination and Fib genes transfer of the plasmid of E . herbicola CSH 1065}; Zhao Y et al.; We inserted kanamycin resistance (Km) gene and mobilized function (Mob) gene on the plasmid of E . herbicola CSH1065 by using DNA molecular cloning and genetic recombination techniques . Therefore, the plasmid of E . herbicola CSH1065 to E . coli HB101 could be transferred by conjugation . The expression of those genes concerning fungi inhibition function (Fib) of E . herbicola CSH1065 in E . coli HB101 was observed . This result confirmed again the fungi inhibition genes of E . herbicola CSH1065 is only related to its plasmid genome not to its chromosome genome . Meanwhile those yellow pigment genes located on the plasmid don't involve the antifungi function of E . herbicola CSH1065 . All those results were convinced by DNA molecular hybridizations.

Acta Vet Scand, 1992, 33(1), 1 - 8
Pathophysiology of experimental bovine endotoxicosis: endotoxin induced synthesis of prostaglandins and thromboxane and the modulatory effect of some non-steroidal anti-inflammatory drugs; Jarlov N et al.; Endotoxin-induced synthesis of thromboxane A2 (TXA2), prostacyclin (PGI2) and prostaglandin E2 (PGE2) was studied in 3 cows after intravenous E . coli endotoxin (055:B5-0.025 mg/kg b.w.) administration . Blood sampling and monitoring of clinical signs were performed from 2 h prior to until 6 h after endotoxin challenge . Blood samples were analyzed for stable hydrolysis products of TXA2 (TXB2), PGI2 (6-keto PGF) and PGE2 (bicyclic PGE2), biochemical and haematological parameters . In a similar experimental design the efficacy of the non-steroidal anti-inflammatory drugs (NSAID) flunixin meglumine (FM) and phenylbutazone (PB) in suppressing eicosanoid synthesis and clinical signs in response to endotoxin challenge was investigated . Two groups of cows, each comprising 2 animals, were treated with FM and PB prior to endotoxin challenge . It was observed that plasma concentrations of TXB2, 6-keto PGF and bicyclic PGE2 increased rapidly after endotoxin challenge . Concentrations were significantly elevated for hours and were correlated to the severity of clinical signs of endotoxicosis . Pretreatment with NSAID suppressed mediator production and alleviated clinical signs . The experiments suggest a certain pathophysiological role of TXA2, PGI2 and PGE2 for the early systemic ill-effects of bovine endotoxicosis.

Vet Res Commun, 1992, 16(1), 59 - 67
Influence of indomethacin on endotoxin-induced changes in gastrointestinal myoelectrical activity and some haematological and clinical parameters in the conscious piglet; De Saedeleer V et al.; The effect of indomethacin, administered intravenously at 5 mg/kg, on the changes in gastrointestinal myoelectrical activity, rectal body temperature, clinical appearance and some haematological parameters induced by intravenous bolus injection of endotoxin, at 10 micrograms/kg, was examined in conscious piglets with electrodes implanted in the antrum pylori, duodenum, jejunum and ileum . Indomethacin inhibited the endotoxin-induced febrile response and the accompanying clinical signs . However, it was without influence on the induced leukopenia and shift to the left . Indomethacin both delayed the onset of and shortened the endotoxin-induced increase in the duration of the antral inhibitory phase and the duodenal phase I activity . It therefore appears that prostanoids are probably not the main factors involved in the endotoxin-induced haematological and gastrointestinal myoelectrical activity changes in the piglet.

Life Sci, 1992, 50(24), 1869 - 76
Contrasting effects of misoprostol on systemic and intrapulmonary lipopolysaccharide-induced tumor necrosis factor-alpha; Nakamura C et al.; Tumor necrosis factor-alpha (TNF), a cytokine produced by mononuclear phagocytes in response to lipopolysaccharide stimulation, is a potent mediator of the inflammatory cascade . However, the immunomodulatory signals regulating TNF expression in the host are poorly defined . Recently, metabolites of the prostaglandin E series have been shown to inhibit TNF production in vitro . In order to determine if PGE1 alters TNF activity in vivo, rats were given misoprostol, a synthetic PGE1 analogue, or saline by gavage and subsequently challenged with either intravenous or intratracheal Escherichia coli lipopolysaccharide . These in vivo data show that PGE1 is a potent inhibitor of TNF production by systemic mononuclear phagocytes . In contrast, alveolar macrophages appear to be refractory to misoprostol's suppressive effects on LPS-induced TNF . This study supports in vitro observations that mononuclear phagocytes within different compartments exhibit differential responsiveness to immunomodulators.

Carbohydr Res, 1992 Jan, 223, 243 - 53
C-glycosyl compounds bind to receptors on the surface of Escherichia coli and can target proteins to the organism; Bertozzi C et al.; A series of C-mannopyranosyl derivatives have been synthesized and their inhibitory activity towards the receptor-mediated adhesion of E . coli to yeast cells has been tested . Total inhibition of yeast-cell agglutination by C-glycosyl derivatives 4 and 9 is achieved at a concentration approximately one order of magnitude lower than that of methyl alpha-D-mannopyranoside, indicating that the binding affinity to the receptor is related to the hydrophobicity of the carbon-linked side chain . A biotin-linked C-glycosyl derivative of mannose (compound 9) has been synthesized and used to target avidin and streptavidin to the bacterial cell surface . Of the C-glycosyl derivatives tested in our study, the conjugate of compound 9 with avidin had the highest avidity for the bacterial receptors, inhibiting agglutination at a concentration three orders of magnitude lower than methyl alpha-D-mannopyranoside . The use of such bifunctional compounds as the mannose-biotin conjugate 9 is a general strategy to target molecules to pathogenic organisms via their cell-surface carbohydrate receptors and to change the antigenicity of the bacterial cell surface.

Anal Biochem, 1992 Jan, 200(1), 163 - 70
A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily--isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain; Banner CD et al.; An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand . Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma . The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds . The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein . Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry . This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.

Anal Biochem, 1992 Jan, 200(1), 143 - 8
Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease; Tyagi SC et al.; A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed . A fluorogenic compound, N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate . The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-beta-naphthylamide) . Fluorescence due to the release of Pro-MeO-beta-naphthylamide was measured continuously by spectrofluorometry . This oligopeptide was found to be a good substrate for HIV-1 protease . The Km and kappa cat for the hydrolysis of N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta- naphthylamide by HIV-1 protease were calculated to be 2.0 +/- 0.2 mM and 75 +/- 6 s-1, respectively . These values are comparable with those of other natural substrates of HIV-1 protease . The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.

J Oral Pathol Med, 1992 Jan, 21(1), 21 - 5
Increased production of tumour necrosis factor by peripheral blood leukocytes in patients with recurrent oral aphthous ulceration; Taylor LJ et al.; Much evidence suggests that recurrent oral aphthous ulceration (RAU) is an immunologically mediated disease . Tumour necrosis factor has multiple biologic properties, some of which may be relevant to the pathogenesis of RAU . This study has assessed its production by peripheral blood leukocytes from aphthous patients in active and remission phases of disease and from patients with nonaphthous ulceration (diseased controls) . Each ulcer patient was studied in parallel with a matched healthy control volunteer . A bioassay against the standard mouse fibrosarcoma line, L929, was used to assess the levels of tumour necrosis factor-alpha (TNF) . Significantly greater amounts of TNF were released from unstimulated monocyte-enriched and monocyte-depleted leukocyte fractions in active RAU compared with those from healthy control donors, suggesting that this cytokine may be associated with RAU.

Gen Pharmacol, 1992 Jan, 23(1), 71 - 4
Effects of nicardipine in rats subjected to endotoxic shock; Lee HC et al.; 1 . The calcium channel blocker, nicardipine, produced a dose-dependent reduction in the mortality caused by endotoxin in rats . 2 . The drug also reduced most of the hematological and gross pathological manifestations of disseminated intravascular coagulation (DIC) caused by endotoxin . 3 . The endotoxin-induced monocytopenia but not the granulocytopenia, lymphocytopenia or thrombocytopenia was inhibited by the drug . 4 . The results suggest that the protective action of nicardipine is causally related to prevention of the endotoxin-induced DIC and that an effect of the drug on monocytes may be of importance.

Dev Biol Stand, 1992, 74, 341 - 51
Lyophilization cycle development for interleukin-2; Vemuri S; The effects of temperature and pressure variation on the lyophilization of interleukin-2 (IL-2) were studied . The human recombinant IL-2 used in this study was synthesized in and purified from E . coli, formulated, and then submitted to experimental lyophilization procedures . The collapse temperature of the formulation was first determined to be -25 degrees C by differential scanning calorimetry . The effects of chamber pressure and shelf temperature on IL-2 during lyophilization were evaluated . Lyophilization chamber pressures were varied from 100 mu to 300 mu in combination with the different chamber pressures . Lyophilized cake quality was assessed by evaluating three of its properties: residual moisture, by the Karl Fischer method; the monomeric content of the protein, by RP-HPLC; and oligomeric content, by SDS-PAGE . Process uniformity was checked by determining residual moisture in cakes collected from various locations in the chamber . The experimental data show that IL-2 can be lyophilized in a pilot unit within 30 hours . The IL-2 lyophilized at various primary drying conditions retained its purity and potency throughout the stability study period (12 months).

Dev Biol Stand, 1992, 74, 295 - 303; discussion 303-6
Development of a lyophilized formulation of interleukin-2; Hora MS et al.; Native human interleukin-2 (IL-2) comprises a group of glycoproteins of MW 13,000-17,500 . Recombinant human IL-2 (rhIL-2) (Cetus) is derived from E . coli and is not glycosylated . We have evaluated several processes for manufacturing rhIL-2, based on different chaotropic agents for solubilization of insoluble protein pastes . Formulation work carried out with material purified by one of these processes is reported here . Our studies have indicated that the presence of a stabilizer in the form of an amorphous excipient, such as amino acids, a non-ionic surfactant (polysorbate 80), hydroxypropyl-beta-cyclodextrin or human serum albumin was essential for preservation of rhIL-2 during lyophilization . Each of these formulations exhibited its own unique problems . We have overcome these problems through a systematic formulation development program and have been successful in developing several lyophilized formulations of rhIL-2 with optimum properties and performance.

Eur J Appl Physiol Occup Physiol, 1992, 64(4), 318 - 22
Changes in blood cell response following strenuous physical exercise; Kvernmo H et al.; The generation of tumour necrosis factor (TNF) and tissue factor activity in lipopolysaccharide (LPS) stimulated blood were studied in 25 healthy subjects before and after physical exercise of different intensities . Of the subjects a group of 9 were athletes who trained once to twice every day of the week, a second group of 8 exercised 3-7 times a week, and a third group of 8 exercised 4-5 times a month . The production of TNF in freshly drawn LPS stimulated blood in heparin, drawn from top athletes at rest was significantly lower than in the other subjects . The LPS induced concentrations of TNF-alpha of 2.73 (SEM 1.05) ng.ml-1 in the blood of the top athletes compared to 5.08 (SEM 0.7) ng.ml-1 and 7.6 (SEM 1.6) ng.ml-1, respectively, in the other two groups . The group that trained the least had the highest values . Immediately after exercise, the monocytes appeared to be less responsive to LPS stimulation, as a reduction of 47%-48% was observed in the top athletes and in the other group of well-trained individuals . The group that trained the least, which was also subjected to the least stressful exercise, had a 33% reduction in TNF production . Within 6 h the TNF concentration was back to pre-exercise values . Within 6 h the TNF concentration was back to pre-exercise values.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Cancer, 1992, 28(2-3), 470 - 3
Phase I study of subcutaneously-administered bacterially-synthesised recombinant human granulocyte-macrophage colony-stimulating factor; Schwartz GK et al.; A phase I study was initiated to test the effect of bacterially-synthesised recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) . 15 patients with advanced cancers were entered into the study and 14 were evaluable . Patients were administered a single subcutaneous injection (3.0-300 micrograms/m2) of rhGM-CSF . Starting at a concentration of 100 micrograms/m2, an approximate 2-fold increase in leucocyte count was noted 24 h after the injection . By 48 h the counts had returned to baseline . The 300 micrograms/m2 concentration also induced an approximate 2-fold increase . The leucocytosis was associated with a predominant increase in circulating neutrophils and bands . An increase in monocytes was also noted, but peak levels were recorded 48-72 h after the injection . At both the 100 micrograms/m2 and the 300 micrograms/m2 doses, significant levels of circulating rhGM-CSF were detected . The levels measured in the plasma of patients receiving 300 micrograms/m2 were over 10-fold greater than those measured at 100 micrograms/m2 . There was no detectable antibody formation against the rhGM-CSF in any of the study patients . The drug was exceptionally well-tolerated . This study shows that rhGM-CSF can be safely administered by subcutaneous administration and a single injection is capable of inducing a leucocytosis with increased circulating neutrophils, bands, and monocytes when doses are used which result in significant levels of circulating rhGM-CSF.

Urol Int, 1992, 48(3), 358 - 61
Prostatic abscess drainage: clinical-sonography correlation; Granados EA et al.; Three cases of prostatic abscess are presented, clinically diagnosed, and verified through transrectal sonography . The diagnostic methods and treatment performed are commented on.

Acta Haematol, 1992, 87(1-2), 22 - 8
Phase I/II study of recombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy . The Multicenter Study Group; Miwa S et al.; The toxicity and hematologic effects of Escherichia coli-derived recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) were studied in 58 treatment cycles in Japanese patients with advanced malignancy as a phase I/II clinical trial . rhGM-CSF in doses from 30 to 250 micrograms/m2/day was administered by 24-hour continuous intravenous infusion, 8-hour intravenous, or a daily subcutaneous injection for 14 days . The most common adverse drug events (ADE) were fever, nausea/vomiting, diarrhea, skin eruption, and phlebitis . The frequency of moderate and severe ADE was 2.9, 14.7, 35.3 and 47.1% at 30, 60, 125, 250 micrograms/m2/day, respectively . In terms of administration routes, the frequency of ADE was 69% with 24-hour continuous intravenous infusion, 39.1% with 8-hour intravenous infusion and 16.7% with subcutaneous injection . Regarding the hematologic effects of rhGM-CSF, leukopenia improved in a dose-dependent manner . The appropriate dose level to be used in the phase II study was estimated to be in the range between 60 and 250 micrograms/m2/day.

Trends Biochem Sci, 1992 Jan, 17(1), 7 - 12
Closed circular DNA as a probe for protein-induced structural changes; White JH et al.; Biological systems are replete with examples of DNA formed into a closed loop structure, either alone or in close association with proteins . Such closed circular DNA molecules are subject to a topological constraint that modifies, often in a major way, the structure and reactivity of the DNA . The topological constraint also permits closed circular DNAs to be used as analytical tools to learn about the structure of DNA-protein complexes.

Toxicology, 1992, 72(3), 341 - 50
Involvement of glutathione in cis-platinum toxicity in Escherichia coli K12; Salles B et al.; Among the various biochemical functions assumed by the tripeptide glutathione (GSH), a role in cell protection against xenobiotics has been well established . In the case of resistance to cis-diamminedichloroplatinum(II) (CDDP) this role is controversial . CDDP reacts with nucleophiles and binds covalently to DNA, its ultimate target . We addressed the question of a putative role of GSH as a secondary non-essential target by using a bacterial model . With an Escherichia coli K12 mutant devoid of GSH, we found sensitivity to CDDP increased by a factor of two . It appeared that GSH protects bacteria at least by covalently trapping platinum before its binding to DNA since (i) lower binding of CDDP to DNA was found when GSH was present and (ii) the resistance still persisted in bacteria after treatment by the monofunctional derivative {Pt(dien)Cl}Cl . On the other hand, with a DNA repair defective mutant (lexA3), we found that other biochemical secondary target(s) might be involved in bacterial protection at low CDDP concentrations.

Microbiol Immunol, 1992, 36(1), 105 - 11
A unique monoclonal antibody that recognizes mature p17 of HIV-1 but not its precursor; Saitoh A et al.; The entire and partial gag regions of human immunodeficiency virus type 1 (HIV-1) were overproduced in Escherichia coli and used for epitope mapping of antibodies against p17 . We found that a mouse monoclonal antibody to p17, V17 recognizes the mature p17 but not the unprocessed Gag proteins containing the entire p17 moiety . Further analysis revealed that V17 recognizes the C-terminal 12-amino-acid region of p17 having free C-terminus . This monoclonal antibody may be useful for monitoring the maturation of virus particles.

Izv Akad Nauk SSSR Biol, 1992 Jan-Feb, (1), 42 - 51
{The effect of the physiological state of Escherichia coli on the nucleoid ultrastructure under stress exposures}; Tkachenko AG et al.; The energetic state and activity of polyamine-synthesizing system of E . coli were studied under conditions of aerobic-anaerobic and pH transitions, nutrition shifts, osmotic and heat shocks . The electron microscopy of cells showed a correlation between cell physiological state and nucleoid ultrastructure . The role of energetic status and polyamines in the regulation of DNA supercoiling is discussed which is a putative cause for the changes observed in the nucleoid ultrastructure.

Biophys Chem, 1992 Jan, 42(1), 7 - 11
Entropies of coding and noncoding sequences of DNA and proteins; Lauc G et al.; The entropies of protein coding genes from Escherichia coli were calculated according to Boltzmann's formula . Entropies of the coding regions were compared to the entropies of noncoding or miscoding ones . With nucleotides as code units, the entropies of the coding regions, when compared to the entropies of complete sequences (leader and coding region as well as trailer), were seen to be lower but with a marginal statistical significance . With triplets of nucleotides as code units, the entropies of correct reading frames were significantly lower than the entropies of frameshifts +1 and -1 . With amino acids as code units, the results were opposite: Biologically functional proteins had significantly higher entropies than proteins translated from the frameshifted sequences . We attempt to explain this paradox with the hypothesis that the genetic code may have the ability of lowering information content (increasing entropy) of proteins while translating them from DNA . This ability might be beneficial to bacteria because it would make the functional proteins more probable (having a higher entropy) than nonfunctional proteins translated from frameshifted sequences.

Biochem Cell Biol, 1992 Jan, 70(1), 63 - 9
Surface denaturation of proteins: the thermal inactivation of beta-galactosidase (Escherichia coli) on wall-liquid surfaces; Edwards RA et al.; Irreversible inactivation of dilute beta-galactosidase (Escherichia coli) at relatively low temperatures was found to occur as a result of interactions of beta-galactosidase with wall-liquid surfaces . The rate of inactivation was directly proportional to the wall-liquid surface area, but independent of the air-liquid surface area, and the rate was also dependent on the wall composition . A small portion of the beta-galactosidase molecules was found to bind strongly to the surfaces of vessels in which the beta-galactosidase was stored . Bovine serum albumin eliminated the inactivation and it also eliminated the binding of beta-galactosidase to the wall . On the other hand, EDTA eliminated the inactivation, but it did not decrease the amount of beta-galactosidase bound . The addition of some transition metals increased the rate of inactivation . Protection of beta-galactosidase from surface inactivation by EDTA is not, therefore, a result of decreased binding of the enzyme to the walls of the vessels, but is probably a result of the ability of EDTA to scavenge certain trace metal ions present in solution, which are needed for the inactivation . The content of protein in the solution did not change as a result of the inactivation and, thus, the inactive enzyme does not accumulate at the surface . Since beta-galactosidase is often stored for long periods of time and since it is used to decrease the lactose content of milk for lactose intolerant individuals, this study may have practical significance . The presence of metal chelators and extraneous proteins should improve the stability of the enzyme, especially for processes that are carried out at elevated temperatures.

Biochem Cell Biol, 1992 Jan, 70(1), 43 - 8
Use of {1-14C}oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity; Ghosh SS et al.; Autoclaved Escherichia coli labelled with {1-14C}oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro . The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity . Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of {1-14C}oleate labelled, autoclaved E . coli optimally at pH 7.0-8.0 to generate {14C}phosphatidic acid in the presence of 5 mM added Ca2+ . Other divalent cations would not substitute for Ca2+ . Activity was linear with time and protein up to 30% of the hydrolysis of substrate . Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100 . The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E . coli by human synovial fluid phospholipase A2 . Accumulation of {14C}diglyceride was observed after 10 min of incubation . This accumulation was inhibited by NaF (IC50 = 18 microM) or propanolol (IC50 = 180 microM) suggesting the S . chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase . Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner . These results demonstrate that {1-14C}oleate labelled, autoclaved E . coli can be used to measure phospholipase D activity by monitoring accumulation of either {14C}phosphatidic acid or {14C}phosphatidylethanol.

Sci China B, 1992 Jan, 35(1), 84 - 91
A new human interferon-gamma mutant harboring EGF receptor-interfering sequence possesses high antiproliferative activity; Wang XM et al.; An interferon gamma (IFN-gamma) mutant gene harboring an additional epidermal growth factor (EGF) sequence at its 3' terminal was constructed and highly expressed in E . coli by using pBV220 as a vector . The new fusion protein-IFN-gamma-EGF possesses not only high antiviral activity, but also high antiproliferative effect as demonstrated by 3H-TdR incorporation method using CN II cells which are rich in EGF receptors.

Ultramicroscopy, 1992 Jan, 40(1), 33 - 53
Three-dimensional reconstruction of single particles embedded in ice; Penczek P et al.; Single particles embedded in ice pose new challenges for image processing because of the intrinsically low signal-to-noise ratio of such particles in electron micrographs . We have developed new techniques that address some of these problems and have applied these techniques to electron micrographs of the Escherichia coli ribosome . Data collection and reconstruction follow the protocol of the random-conical technique of Radermacher et al . {J . Microscopy 146 (1987) 113} . A reference-free alignment algorithm has been developed to overcome the propensity of reference-based algorithms to reinforce the reference motif in very noisy situations . In addition, an iterative 3D reconstruction method based on a chi-square minimization constraint has been developed and tested . This algorithm tends to reduce the effects of the missing angular range on the reconstruction, thereby facilitating the merging of random-conical data sets obtained from differently oriented particles.

Development, 1992 Jan, 114(1), 271 - 83
Lineage of radial glia in the chicken optic tectum; Gray GE et al.; In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation . To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E . coli lacZ gene . The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product . Radial glia were identified in a substantial fraction of clones, and these were studied further . Our main results are the following . (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia . Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor . (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7) . Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce . (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia . Thus, radial glia may frequently guide the migration of their clonal relatives . (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes . This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching . To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.

Vet Q, 1992 Jan, 14(1), 29 - 34
The pathogenesis of the post-weaning syndrome in weaned piglets: a review; van Beers-Schreurs HM et al.; This review deals with the pathogenesis of the post-weaning syndrome . This syndrome includes post-weaning diarrhoea (PWD), oedema disease (OD) and endotoxin shock (ES) . The role of different enteropathogenic Escherichia coli bacteria and some other predisposing factors relating to this post-weaning syndrome (PWS) are discussed . Based on intestinal pathophysiological mechanisms, some suggestions for the prevention of PWS and prospects for future research are given.

Life Sci, 1992, 50(19), 1459 - 68
Effect of the aromatase inhibitor, 4 hydroxyandrostenedione, on the endotoxin-induced changes in steroid hormones in male rats; Christeff N et al.; The increase in circulating estrogen concentrations that follows injection of Escherichia coli endotoxin (Endo) may be due to increased aromatase activity . We have therefore analysed the effect of the aromatase inhibitor, 4 hydroxyandrostenedione (4OHA) on the steroid hormone response of male rats, particularly the dramatic increase in estrogens and decrease in androgens, induced by Endo . The concentrations of corticosterone (B), progesterone (P4), 17 alpha hydroxyprogesterone (17 alpha OHP4), androstenedione (delta 4), testosterone (T), estrone (E1) and estradiol (E2) were determined 2 hours after injection of increasing doses of 4OHA with and without Endo . The increase in serum estrogen concentrations and drop in serum androgen levels in response to Endo were blocked by a single dose of 4OHA . The effect of 4OHA appeared to be dose dependent . Low doses (30 mg/kg and 50 mg/kg) induced significant changes in the estrogen and androgen responses, but the high dose (100 mg/kg) blocked all changes in sex steroids induced by Endo . 4OHA did not alter the Endo-induced changes in other steroids.

Eur J Epidemiol, 1992 Jan, 8(1), 74 - 80
Chinese hamster ovary cells produce an enzyme that nicks heat-labile enterotoxin from enterotoxigenic Escherichia coli; Tsuji T et al.; Chinese hamster ovary (CHO) cells were examined for production of an enzyme that nicked the polypeptide chain of the heat-labile enterotoxin from enterotoxigenic Escherichia coli between the A1 and A2 fragments of its A subunit . Serum-free culture medium prepared each day after CHO cell inoculation was concentrated 100 times and its proteolytic activity for formation of the A1 fragment was examined by Western blotting with anti-LT A antibody . The A subunit was detected in culture medium on day 6 after cell inoculation, although not in media on day 1 or 3, indicating that CHO cells produced a nicking enzyme . This nicking enzyme had an optimal pH of about 7.5 and an apparent Mr . of 120,000, as seen by Superose 12 TM gel filtration with an FPLC system . The activity of this enzyme was strongly inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by p-chloromercuribenzoic acid, EDTA or ethyleneglycol bis (beta-aminoethylether)-N,N-N',N'-tetraacetic acid, suggesting that this enzyme was a serine protease . The activity was not stimulated by plasminogen or fibrin . These findings suggest that the nicking enzyme was different from proteases such as elastase, collagenase and plasminogen activator, which are probably also secreted by fibroblast-like CHO cells.

Eur J Nucl Med, 1992, 19(3), 166 - 72
Regional perfusion, oxygen metabolism, blood volume and immunoglobulin G accumulation at focal sites of infection in rabbits; Senda M et al.; Infection causes remarkable changes in extracellular fluid volume, blood flow and oxygen consumption in the region of the lesion . To determine the sequence and magnitude of these changes, we performed serial scintigraphic measurements in 10 rabbits with experimental Escherichia coli abscesses . Positron emission tomography with C15O2, 15O2 and 11CO was used to measure regional blood flow, oxygen extraction (OEF) and blood volume; extracellular fluid volume was evaluated by single photon scintigraphy with indium-111 immunoglobulin G (IgG) . Images were recorded following tracer administration at 1 and 7-10 days after infection . At the first imaging time, blood flow to infected muscle had increased by 40% compared with control sites (7.4 +/- 0.6 to 10.8 +/- 3.8 ml/min.100 g), OEF had decreased from 55% +/- 34% to 45% +/- 14%, and the infected-to-contralateral (I/C) ratio of IgG had increased to 3.34 +/- 1.85 . At the later imaging time, flow had increased by almost threefold compared with day 1 (29.4 +/- 9.8 ml/min.100 g), OEF had decreased to 29% +/- 14%, and the I/C ratio for IgG had remained constant . Although OEF fell, oxygen delivery (OEF x flow) increased from 4.07 ml/min (control value) to 4.86 ml/min on day 1 and 8.64 ml/min on days 7-9 . The infected-to-contralateral (IC) ratio of 15O2/C15O2 was 0.74 +/- 0.15 on day 1 and 0.77 +/- 0.10 at 7-9 days . These studies indicate that expansion of the extracellular fluid volume increases early in the evolution of the infection and exceeds changes in regional perfusion and oxygen delivery.

Eur J Nucl Med, 1992, 19(3), 159 - 65
Accumulation of immunoglobulin G at focal sites of inflammation; Juweid M et al.; To evaluate the factors responsible for the accumulation of indium-111 immunoglobulin G (111In-IgG) at sites of inflammation, sequential measurements of tissue blood volume, interstitial fluid volume and accumulation of radiolabelled albumin and IgG were made in rats following Escherichia coli infection in the thigh . Compared with normal thigh muscle, there was approximately two-fold increase in interstitial fluid volume and approximately 1.5-fold increase in plasma and red blood cell volumes in infected muscle . For both proteins, there was a fivefold increase in influx rate constant (kin) in infected muscle . In normal muscle, the interstitial fluid concentration of labelled human serum albumin (111In-HSA) was significantly higher than that of 111In-IgG (P less than 0.01) . In contrast, the concentrations in infected muscle were nearly identical . The concentration ratios (infected to normal muscle) were 1.7:1 for HSA and 3:1 for IgG . These data suggest that the infection imaging properties of 111In-IgG are related to expansion of the space available to macromolecules in infected tissue and increased transport into this space . At clinically important imaging times (24-48 h after injection), the higher target-to-background ratio of 111In-IgG compared with 111In-HSA is not due to the higher accumulation IgG in infected tissue but rather to the higher accumulation of HSA in normal tissue.

Anticancer Res, 1992 Jan-Feb, 12(1), 135 - 9
Antiplasmid activity of phenothiazines, benzo{a}phenothiazines and benz{c}acridines; Motohashi N et al.; Various synthetic derivatives of phenothiazines, benzo{a}phenothiazines and benz{c}acridines were compared for their abilities to induce antiplasmid activity against E . coli F'lac plasmid . Several phenothiazine derivatives were much more potent in antiplasmid activity than benzo{a}phenothiazine- or benz{c}acridine derivatives . Their antiplasmid activity seemed to be enhanced by Cl- or CF3- substitution at 2 C atom, and modified by the side chain length and charge at the L-region of the molecules, as well as by hydrophilicity.

J Struct Biol, 1992 Jan-Feb, 108(1), 74 - 89
The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins; Heitlinger E et al.; Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain . To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered "headless," "tailless," and "rod" chicken lamin B2 cDNAs and expressed them in Escherichia coli . A full-length chicken lamin A cDNA was also expressed in E . coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E . Heitlinger et al . (1991) J . Cell Biol . 113, 485-495) . As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end . Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod . In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E . Heitlinger et al . (1991) J . Cell Biol . 113, 485-495), associated longitudinally to form polar head-to-tail polymers . Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association . Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.

Vaccine, 1992, 10(4), 217 - 20
Enhancement of the immune response by intradermal vaccination in cattle with enterotoxigenic Escherichia coli (ETEC) vaccine without adjuvant; Itzchak S et al.; A comparison was made of the efficiency of intradermal and subcutaneous routes of vaccination in immunizing cattle with a killed vaccine of Escherichia coli K99+ . In this study 135 heifers aged 8-18 months were included . The effect of the number of intradermal injection sites was examined, as well as the optimal time interval between the initial and booster intradermal vaccination . The intradermal route was found to be significantly superior to the subcutaneous route . The optimal time for intradermal booster was approximately 2 weeks . Increasing the number of intradermal injection sites up to 50 (using the same amount of antigen) did not result in an enhancement of the antibody level.

Microb Pathog, 1992 Jan, 12(1), 47 - 62
Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp60 protein, BA60K; Roop RM 2nd et al.; A recombinant 60 kDa Brucella abortus protein expressed in Escherichia coli was recognized in immunoblots by sera from mice experimentally infected with B . abortus and a dog experimentally infected with B . canis . Sera from humans and dogs with naturally acquired brucellosis also recognized this protein, which was designated BA60K . The gene encoding BA60K was localized within an 18 kb B . abortus genomic fragment and its direction of transcription determined by subcloning and maxicell analysis of selected restriction fragments . The nucleotide sequence of 1800 bases encompassing the predicted gene location was determined, revealing an open reading frame encoding a protein of 546 amino acids (predicted relative molecular mass of 57515) . Solid phase micro-sequencing of BA60K eluted from two-dimensional polyacrylamide gels confirmed the predicted amino acid sequence . Comparison of the predicted amino acid sequence of BA60K with a protein sequence database revealed that BA60K shares 67.9% identity with the GroEL protein of E . coli, a member of the Hsp60 family of chaperonins . The immunodominant Hsp60 homologs from Legionella pneumophila, Chlamydia trachomatis and Mycobacterium tuberculosis were also found to share greater than 59% amino acid sequence identity with the BA60K protein . The identification of BA60K as a member of the Hsp60 family of chaperonins supports its role in stimulating a prominent host immune response during the course of Brucella infections . It also indicates that BA60K is an important candidate for studies aimed at identifying the antigens responsible for eliciting the protective immune response to brucellosis.

Zentralbl Bakteriol, 1992 Jan, 276(2), 254 - 63
Surface properties, connective tissue protein binding and Shiga-like toxin production of Escherichia coli isolated from patients with ulcerative colitis; Olusanya O et al.; Escherichia coli strains isolated from intestinal biopsies of patients with ulcerative colitis (n 146), Crohn's disease and colonic polyposis (n 41) were analysed for binding of collagen I, collagen IV, fibronectin and laminin . Strains expressed varying degrees of binding of one or more of the four connective tissue proteins . Only 32 strains did not express binding of any of the proteins . The strains expressed low or moderate cell surface hydrophobicity . There was no correlation between protein binding and expression of cell surface hydrophobicity . E . coli isolated from inflamed rectal mucosa were slightly less negatively charged than strains isolated from healthy intestinal mucosa . Shiga-like toxins I and II were detected in 32 strains from 28 patients . Of these, 5 strains had been isolated from normal or healed tissue . In patients with inflammatory bowel disease, connective tissue proteins are exposed in intestinal ulcerations . Strains expressing binding of one or several of these proteins may have a selective advantage to colonize these lesions.

Zentralbl Bakteriol, 1992 Jan, 276(2), 231 - 42
Adherence to and cytotoxicity of Escherichia coli for eucaryotic cell lines quantified by MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide); Kreft B et al.; Adherence of Escherichia coli to human epithelial cells (HEp-2) was studied using MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) which is cleaved by enzymes of eucaryotic or procaryotic cells to formazan . This method allows to quantify adherence of Escherichia coli to HEp-2 cells and offers the advantage of assaying a large number of eucaryotic cells without using specific antisera or radioactive material . Furthermore, toxic effects of isolated hemolysin cloned in Escherichia coli onto a renal tubular cell line (LLC-PK1) was investigated by this method, showing reduced cellular viability of tubular cells after an incubation period of 10 to 20 min . MTT is therefore considered to be useful to assay the adherence of Escherichia coli to eucaryotic cells and to quantify toxic effects in eucaryotic cells induced by bacterial virulence factors.

Zentralbl Bakteriol, 1992 Jan, 276(2), 196 - 204
Core-lipid A on the K40 polysaccharide of Escherichia coli O8:K40:H9, a representative of group I capsular polysaccharides; Jann K et al.; From the capsular K40 polysaccharide of E . coli O8:K40:H9, a fraction was obtained by gel permeation chromatography which in SDS-PAGE exhibited a ladder-like pattern characteristic of lipopolysaccharides . In Western blots, this fraction reacted with a K40-specific antiserum but not with an O8-specific antiserum . It contained, in addition to the constituents of the K40 polysaccharide (glucuronic acid, glucosamine and serine), glucose, galactose, heptose, and KDO . Mild acid hydrolysis of this fraction liberated a lipid moiety which by chemical analysis was characterized as lipid A . From these results, we conclude that the capsular polysaccharide of E . coli O8:K40:H9 is in part bound to core lipid A . The significance of this finding is discussed.

Zentralbl Bakteriol, 1992 Jan, 276(2), 165 - 75
Genetics of Escherichia coli uropathogenicity: analysis of the O6:K15:H31 isolate 536; Hacker J et al.; E . coli strain 536 (O6:K15:H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins . The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain . Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode . Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E . coli K-12 linkage map . By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated . In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate . This particular mode of virulence regulation is missing in the mutant strain.

Microbiologica, 1992 Jan, 15(1), 1 - 6
Plasmid analysis in PPNG strains isolated in Italy; Manicardi G et al.; Italian reports of PPNG strains are rare . In this paper we report the plasmidial characteristics of two strains: one harbouring 3.2 and 2.6 Mdal plasmids and the other 24.5, 4.5 and 2.6 Mdal plasmids . Restriction analysis was carried out on plasmids transferred to E . coli by transformation for "Africa" and conjugation for "Asia" plasmid.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 17 - 21
Rate of plasmid transfer among Escherichia coli strains isolated from natural populations; Gordon DM; The conjugative plasmid R1 was introduced into ten strains of Escherichia coli isolated from natural populations . Spontaneous nalidixic-acid-resistant mutants of the ten strains served as recipients . The ten donor and recipient strains were mated in all combinations and the rate at which R1 transferred between the strains was determined . The rate of transfer ranged from 5.2 x 10(-11)-1.1 x 10(-18) ml per cell h-1, and averaged 1.3 x 10(-15) ml per cell h-1 . The results of these experiments suggest that the rates of conjugative transfer are far too low for plasmids to be maintained as parasites in their host populations . Infectious transfer is insufficient; plasmids must confer a selective advantage to their host to be maintained.

Fiziol Zh, 1992 Jan-Feb, 38(1), 68 - 72
{The role of the leukocytes in increasing the vascular permeability in an infectious inflammation focus}; Klymenko MO; The model of acute infectious peritonitis in rats with vinblastine-induced leukopenia has been used to show that the leukocyte depletion significantly influences the vascular permeability of abdominal cavity during the whole period of exudation . It inhibits the vascular permeability rise both in the immediate phase and in the initial period of delayed phase (5 h) . 12 hours-5 days after the inflammatory agent action the vascular permeability under conditions of primary leukopenia appears to be more than in the natural course of inflammation, that coincides with excess of the leukocyte number usual for the inflammatory focus and with its significant increase in blood . The results indicate the essential role of leukocytes both during the immediate and delayed phases of increase in the vascular permeability of inflammatory infectious focus.

Fiziol Zh, 1992 Jan-Feb, 38(1), 64 - 8
{Mast cells in the focus of an acute infectious inflammation}; Klymenko MO et al.; On the model of E . coli-induced acute infectious peritonitis in rats it is established that the mast cell reaction and histamine level increase in exudate and inflamed mesentery tissue are biphase and are observed predominantly following the inflammatory agent action, in the period corresponding to the immediate phase of peritoneal cavity vessel permeability increases . The preliminary elimination of mast cells significantly inhibits a rise in the vascular permeability in the immediate phase and slightly affects the delayed phase, thus prolonging exudation . At the same time the dynamics of free histamine indicates its direct involvement in mediation and/or modulation as well as in subsequent inflammatory events . The common rules of mast cell involvement and vascular permeability increase in infectious and aseptic inflammation have been shown.

Life Sci, 1992, 50(16), 1137 - 42
Presence and some characterization of GDP dissociation inhibitors for a low Mr GTP-binding protein, ram p25, in rat spleen cytosol; Nagata K et al.; Two proteinous factors, designated here as ram p25 GDP dissociation inhibitor (I) and (II) (ram-GDI (I) and (II)), were detected in the cytosolic fraction of rat spleen, which inhibited the initial dissociation of GDP from ram p25 produced by E . coli by causing characteristic lag . They had very weak effects on the rate of dissociation of GDP after the lag, and did not affect the mode of the dissociation of 5'-(3-O-thio)triphosphate (GTP gamma S) from ram p25 . By gel filtration, the molecular masses of ram-GDI (I) and (II) were calculated to be 90 KDa and 40 KDa, respectively . These ram-GDIs did not affect the GDP-dissociation of Ha-ras protein produced in E . coli.

Bioelectromagnetics, 1992, 13(1), 75 - 8
Microwave-specific heating affects gene expression; Saffer JD et al.; The effects of low-level microwave radiation on gene expression in Escherichia coli have been examined in a sensitive model . We confirm the previously reported existence of an increase in beta-galactosidase expression by microwave radiation--an increase not duplicated by bulk heating . However, the effect was not frequency dependent and appeared to be due to heating effects peculiar to microwaves . These results indicate that small thermal gradients may be a source of biological effects of non-ionizing radiation.

Arch Virol, 1992, 123(1-2), 111 - 24
Expression in vivo and in vitro of the major structural protein (VP73) of African swine fever virus; Cistue C et al.; The VP73 protein was produced by in vitro transcription and translation from the Xho I-Bam HI fragment located between the Cla I-N and Cla I-H fragments of the viral genome . This DNA fragment encodes a late mRNA of about 2.6 kb detected in infected MS monkey and BHK hamster cells . The transcript was initiated at a site within two bases upstream of the translation initiation codon . The in vitro synthesized polypeptide shows the same molecular weight as the in vivo synthesized polypeptide, suggesting that VP73 has no post-translational modification . There are two internal AUG initiation codons for in vitro translation, one of which is functional in vivo, as well as a possible GUG initiator codon detected by expression of the protein in E . coli cells.

Arch Virol, 1992, 123(1-2), 1 - 11
Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of polyhedrin gene expression; van Oers MM et al.; Two major late proteins, polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells . This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression . To address the question whether the Autographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the polyhedrin gene, several recombinants were constructed with various deletions in the polyhedrin gene . The Escherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and polyhedrin-driven expression . None of the deletions in the polyhedrin gene did result in higher expression of the p10 promoter-controlled gene . This suggested that the transcriptional and/or translational activity of the p10 and polyhedrin gene are independently regulated . To compare the level of polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied . No significant difference in level of expression was observed . In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected . This may be due to instability of the viral genome, as two copies of the lacZ gene were present.

Mol Microbiol, 1992 Jan, 6(2), 239 - 46
A 3' transcriptional enhancer within the coding sequence of a yeast gene encoding the common subunit of two multi-enzyme complexes; Zaman Z et al.; A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different LPD::lacZ gene fusion integrated at the ura3 locus . These LPD::lacZ fusions differ in the amount of the LPD1 gene (encoding lipoamide dehydrogenase) that is fused to the lacZ reporter . Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the LPD1 coding region between +13 and +700 is involved in activating gene expression in a carbon source-dependent manner . This activation occurs at the mRNA level, and is not mediated by changes in mRNA stability . Therefore, the LPD1 gene appears to contain a transcriptional enhancer that lies 3' to the transcriptional start site, and which responds to carbon source.

Mol Microbiol, 1992 Jan, 6(2), 221 - 30
Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli; Blasco F et al.; Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli . Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively . The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme . A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase . When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble . When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble . When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane . It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity . The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme . The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits . Although this corresponds to the definition of a chaperone protein this possibility has been rejected . In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.

Mol Microbiol, 1992 Jan, 6(2), 209 - 19
Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli; Blasco F et al.; Two nitrate reductases, NRA and NRZ, are present in Escherichia coli . These isoenzymes have the same alpha beta gamma, subunits composition and have similar size and genetic organization . Corresponding subunits of the complexes share at least 75% identity . By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the alpha subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane-bound heterologous enzyme . One such heterologous enzyme, alpha A beta Z gamma Z, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis . It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced . The possibility that heterologous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.

Mol Biochem Parasitol, 1992 Jan, 50(1), 47 - 56
Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata; Field H et al.; Trypanothione reductase (TR) is a target for drug design since it is unique to trypanosomatids, substituting for the otherwise ubiquitous enzyme, glutathione reductase . We report the cloning and sequencing of several cDNAs and genes encoding Crithidia fasciculata TR, the structure of which has recently been solved by crystallography . Single base polymorphisms are detected in cDNAs (containing 80% of the coding sequence) and two different genomic clones, including a glutamine to glutamate change in the C-terminal region of the TR coding region; other nucleotide changes are silent . Homology (from genomic clones, both of which contained signals appropriate for expression) to the Trypanosoma congolense gene was 63% at the nucleic acid level, with 68% amino acid identity; the significance of homologies to human and Escherichia coli glutathione reductase sequences is discussed . Polymorphic sites in the genomic clones included sites found in the cDNAs, indicating that differences existing in the genomic sequence are real, and propagated to RNA.

J Photochem Photobiol B, 1992 Jan, 12(1), 9 - 27
Phage nucleoprotein-psoralen interaction: quantitative characterization of dark and photoreactions; Ronto G et al.; The irradiation of the phage T7 system containing psoralen as photosensitizer causes many processes, each of them leading to phage inactivation . These processes include the UV-induced photoreactions in the phage nucleic acid, and photoreactions in the nucleic acid sensitized by either psoralen or psoralen photobreakdown products . In addition the intercalation of the psoralen molecule itself in the phage nucleic acid as well as the psoralen photobreakdown products cause phage inactivation . Under appropriate experimental conditions these reactions can be studied and characterized separately . The quantitative characteristics (e.g . inactivation cross-section, action spectra and index for dark genotoxicity) are demonstrated for different linear and angular psoralens . Some theoretical and practical consequences of the results obtained are discussed.

J Dairy Sci, 1992 Jan, 75(1), 72 - 7
Opsonic activity of bovine serum and mammary secretion after Escherichia coli J5 vaccination; Hogan JS et al.; Six pairs of cows were used to determine the effects of immunization with an Escherichia coli (O111:B4) J5 bacterin on in vitro opsonization of a smooth heterologous strain of E . coli . One cow in each pair was either immunized with the vaccine or sham-immunized at drying off, 30 d after drying off, and at calving . Opsonizing bacteria with serum collected from vaccinated cows 21 d after calving resulted in higher mean number of intracellular bacteria per phagocytosing neutrophil than opsonizing bacteria with serum collected from control cows . Phagocytic parameters using serum collected at drying off and calving did not differ between treatment groups . A trend for enhanced opsonic activity of colostrum from vaccinates was noted . Enhanced opsonization by serum from vaccinated cows coincided with higher serum IgM titer to E . coli J5 whole cell antigen compared with controls . Serum IgG titers to E . coli J5 did not differ between groups . Colostrum IgG titers to E . coli J5 were greater at calving in vaccinated than in control cows . Colostrum and milk collected 21 d after calving from vaccinated cows had higher IgM titers to E . coli J5 than did mammary secretions from control cows . Numbers of intracellular bacteria per phagocytizing neutrophil were correlated positively with IgM titers to E . coli J5 in both serum and colostrum.

Appl Environ Microbiol, 1992 Jan, 58(1), 412 - 4
Lack of virulence factors in Escherichia coli strains of enteropathogenic serogroups isolated from water; Valentini SR et al.; Thirty-eight Escherichia coli strains belonging to 14 human enteropathogenic serogroups were isolated from 33 of 208 water samples studied . No virulence factor or virulence-related gene sequences were found in any of the 38 strains analyzed . The results point out the importance of detecting specific virulence factors before incriminating water as a source of human diarrhea.

Appl Environ Microbiol, 1992 Jan, 58(1), 392 - 8
Biotic and abiotic factors affecting plasmid transfer in Escherichia coli strains; Fernandez-Astorga A et al.; The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated . The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1 . As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10 . The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1 . However, the transconjugants were detected even with less than 1 mg of C liter-1 . Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001) . Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed . We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.

Appl Environ Microbiol, 1992 Jan, 58(1), 331 - 4
Unique and overlapping pollutant stress proteins of Escherichia coli; Blom A et al.; Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis . Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical . The stress protein induction showed a temporal pattern, indicating sequential gene expression . Chemical stress protein synthesis occurred even at concentrations that had no effect on growth . Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.

Poult Sci, 1992 Jan, 71(1), 27 - 37
Replicated divergent selection of broiler chickens for high or low early antibody response to Escherichia coli vaccination; Leitner G et al.; Four sublines of broiler chickens were selected from a base population for three generations for high or low antibody response to vaccination with Escherichia coli at 10 days of age . Two sublines were selected for a high response (HC) and two for a low response (LC) . Realized heritability estimates over three generations of selection for each pair of replicated lines averaged .23 in HC lines and .32 in LC lines . No correlated response in the important production trait, body weight at marketing age, was observed . The ability to survive pathogenic E . coli challenge with or without prevaccination showed no differences between the lines in the unvaccinated chicks, although following vaccination there was higher mortality and morbidity in the LC lines . These data suggest that the use of antibody response in young chicks to E . coli vaccine may be a useful genetic indication of more general disease resistance.

J Appl Physiol, 1992 Jan, 72(1), 259 - 65
Regulation of perfused capillary density in canine intestinal mucosa during endotoxemia; Drazenovic R et al.; When O2 delivery (blood flow X arterial O2 content) is reduced, many tissues respond by increasing perfused capillary density . This facilitates the increase in O2 extraction required to maintain tissue O2 consumption in the face of limited O2 supply . In a previous study of isolated canine small intestine (J . Appl . Physiol . 64: 2410-2419, 1988), endotoxin administration was associated with an impaired ability to increase O2 extraction in response to progressive reductions in O2 delivery . The aim of the present study was to determine whether reductions in perfused capillary density occur after endotoxin administration . Fourteen male dogs were anesthetized with chloralose (150 mg/kg iv) and urethan (750 mg/kg iv), and a segment of small intestine was exteriorized through a midline laparotomy . The segment was isolated vascularly, autoperfused, and maintained at body temperature . Escherichia coli endotoxin (5 mg/kg) or sham challenge was administered, and the animals were allowed to stabilize . Blood flow and arterial and gut venous blood O2 contents were measured after 3 h . Perfused vessels were then labeled by injecting colloidal carbon (less than 0.8 microns) through the arterial cannula and clamping the artery and vein as the bolus passed through the tissue . In some of the experiments a second gut segment was successfully obtained within 1 h of the first, yielding a total of 14 gut segments in nine endotoxin animals and nine segments in five control animals . Morphological analysis of capillary surface density in mucosal villi and crypts showed a significantly higher perfused capillary density in control tissue blocks (77.8 +/- 9.2%) than in blocks from endotoxin-treated animals (68.8 +/- 8.0%, P less than 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1992 Jan 1, 69(2), 141 - 6
Analysis of K99 plasmids from enterotoxigenic Escherichia coli; Isaacson RE et al.; Evaluation of 9 wild-type K99 positive strains of Escherichia coli showed that each had a plasmid of approximately 87.8 kb that hybridized with two DNA probes specific for K99 genes . The K99 reference plasmid from E . coli also is 87.8 kb . Each of these strains had a conserved 7.15-kb BamHI fragment that also hybridized to these probes . Several K99 negative mutants and three 3P- strains also contained K99 plasmids as well as the 7.15-kb BamHI fragment . These results suggest that there is a conservation in size of the K99 plasmids of diverse strains.

Mol Gen Mikrobiol Virusol, 1992, (1-2), 3 - 7
{Proteins, associated with 50S-ribosomal subunits of Escherichia coli MRE600}; Orlov VG et al.; The proteins associated with the ribosomal subunits having the molecular masses from 158 to 47 kDa were isolated from hyaloplasmic, nucleoid and membrane fractions of Escherichia coli MRE600 cells . The proteins are eliminated from 50S subunits of ribosomes by thrice washing with the 1 M ammonium chloride buffer . 50S subunit proteins were found to be immunologically related to the inner membrane proteins . The native 50S subunits of ribosomes possess the expressed ATP-ase activity, while the washed off subunits lose it completely.

Int J Radiat Oncol Biol Phys, 1992, 22(4), 677 - 9
Repair of damage induced by SR 4233; Edwards DI et al.; The benzotriazine di-N-oxide, SR 4233, was electrolytically reduced at constant potential at pH 4.0 at a reduction rate of 5%/hr under N2 in the presence of phi X174 DNA . During the reduction process, the biological infectivity of the bacterial phage was measured by a double transfection technique, either into the wild-type Escherichia coli strain, or into a series of seven mutants with specific, known defects in their capacity to repair DNA . The survival of phi X174 was evaluated as an index of drug damage and from this we conclude that SR 4233 induces pH-dependent DNA damage in E coli, which is recognized and repaired primarily by the uvrC gene product and by the exonuclease III and endonuclease III gene products . These gene products act primarily upon and are responsible for the recognition of strand breaks and repair of oxidized and fragmented pyrimidine products, indicating that SR 4233 induces strand breaks in DNA resulting from oxidative damage to pyrimidines . As damage is maximized at acid pH, we further propose that the damage mechanism is a process of electron transfer from pyrimidine nucleotides in DNA (i.e., oxidation) to the protonated benzotriazine di-N-oxide one-electron radical anion.

Curr Genet, 1992 Jan, 21(1), 43 - 7
Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu-3); Gainey LD et al.; The nucleotide sequences of the genes encoding the acetate-inducible glyoxylate cycle enzyme isocitrate lyase from the ascomycete fungi Aspergillus nidulans (acuD) and Neurospora crassa (acu-3) are presented . The respective A . nidulans and N . crassa genes are interrupted at identical positions by two introns and encode proteins of 538 and 543 amino acids, which have 75% identity . The predicted protein sequences do not demonstrate the C-terminal tripeptide S-K-L that has been implicated in peroxisomal targeting and found in the glyoxysomally located enzyme malate synthase from the same species . However, the protein sequences do exhibit a partial repeat which, in common with malate synthase, is located in regions that are absent from, or non-homologous with, the E . coli enzyme, which is not compartmentalized.

Genetics, 1992 Jan, 130(1), 17 - 26
Mutant lambda repressors with increased operator affinities reveal new, specific protein-DNA contacts; Benson N et al.; The binding specificities of four mutant lambda cI repressor proteins with increased affinities for operator DNA were examined . Two mutant repressors (Glu34----Lys and Glu83----Lys) have the same specificity of binding as wild-type repressor, whereas two (Gly48----Ser and Gly48----Asn) have new binding specificities . The Gly48----Asn mutant repressor recognizes lambda operators with changes at base pair 3 with a different order of affinity than wild-type repressor, suggesting that the side chain of Asn48 makes additional specific DNA contacts at or near this base pair . When paired with a change that disrupts the specific interaction of the amino-terminal arm of lambda repressor with DNA (Lys4----Gln), one change that increases the affinity of repressor (Gly48----Ser) suppresses the binding defect of the Lys4----Gln repressor, resulting in a double mutant repressor with a new binding specificity different than that of both its parents and of wild type . These results lend strong support to the model of direct recognition of the lambda operator by lambda repressor proposed from the crystal structure of the repressor/operator complex.

J Gen Virol, 1992 Jan, 73 ( Pt 1), 17 - 25
Specific binding of influenza A virus NS1 protein to the virus minus-sense RNA in vitro; Hatada E et al.; The non-structural protein NS1, encoded by genome segment 8 of influenza A virus, was expressed in Escherichia coli from cloned cDNA and purified . The NS1 protein had a specific RNA-binding activity, binding to influenza A virus minus-sense but not plus-sense RNA synthesized in vitro from cloned DNA using phage RNA polymerase . NS1 bound preferentially to the regions of RNA containing either 5'- or 3'-terminal common sequences of the genomic RNA . Binding was inhibited by virion RNA, but not by single-stranded minus-sense cDNA and oligo DNAs having the common sequences . In addition, binding was also inhibited by 28S rRNA but not 18S rRNA prepared from MDCK cells.

J Bacteriol, 1992 Jan, 174(1), 331 - 5
Synergism between the Trp repressor and Tyr repressor in repression of the aroL promoter of Escherichia coli K-12; Heatwole VM et al.; Computer analysis identified a potential Trp repressor operator 56 nucleotides downstream of the transcriptional start point of aroL, the gene that encodes shikimate kinase II . Tryptophan-dependent interaction of Trp repressor with this operator was demonstrated in vitro by means of a restriction endonuclease protection assay . Regulation of expression from the aroL promoter was evaluated with several genetically marked Escherichia coli strains by using a single-copy aroL-lacZ transcriptional-translational reporter system . The expression of aroL was repressed 6.9-fold by the Tyr repressor alone and 29-fold when both Tyr and Trp repressors were present . The Trp repressor had no effect on expression from the aroL promoter in the absence of the Tyr repressor . Possible mechanisms for Trp repressor-mediated repression, including cooperative interactions with the Tyr repressor, are discussed.

Arch Biochem Biophys, 1992 Jan, 292(1), 87 - 94
Role of the amino terminal region of the epsilon subunit of Escherichia coli H(+)-ATPase (F0F1); Jounouchi M et al.; Escherichia coli strain KF148(SD-) defective in translation of the uncC gene for the epsilon subunit of H(+)-ATPase could not support growth by oxidative phosphorylation due to lack of F1 binding to Fo (M . Kuki, T . Noumi, M . Maeda, A . Amemura, and M . Futai, 1988, J . Biol . Chem . 263, 17, 437-17, 442) . Mutant uncC genes for epsilon subunits lacking different lengths from the amino terminus were constructed and introduced into strain KF148(SD-) . F1 with an epsilon subunit lacking the 15 amino-terminal residues could bind to F0 in a functionally competent manner, indicating that these amino acid residues are not absolutely necessary for formation of a functional enzyme . However, mutant F1 in which the epsilon subunit lacked 16 amino-terminal residues showed defective coupling between ATP hydrolysis (synthesis) and H(+)-translocation, although the mutant F1 showed partial binding to Fo . These findings suggest that the epsilon subunit is essential for binding of F1 to F0 and for normal H(+)-translocation . Previously, Kuki et al . (cited above) reported that 60 residues were not necessary for a functional enzyme . However, the mutant with an epsilon subunit lacking 15 residues from the amino terminus and 4 residues from the carboxyl terminus was defective in oxidative phosphorylation, suggesting that both terminal regions affect the conformation of the region essential for a functional enzyme.

Virology, 1992 Jan, 186(1), 207 - 18
The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector; Giraud C et al.; An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced . We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence . The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates . Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides . This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence . Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication . Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically . Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions . Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.

J Virol, 1992 Jan, 66(1), 420 - 31
In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules; Cotmore SF et al.; During replication of their linear, single-stranded DNA genomes, parvoviruses generate a series of concatemeric duplex intermediates . We have cloned, into Escherichia coli plasmids, junction fragments from these palindromic concatemers of minute virus of mice DNA spanning both the right end-to-right end (viral 5' to 5') and left end-to-left end (viral 3' to 3') fusions . When mouse cells were transfected with these circular plasmids and superinfected with minute virus of mice, the viral junctions were resolved and the plasmids replicated as linear chromosomes with vector DNA in their centers and viral DNA at their termini . Resolution did not occur when the concatemer joint was replaced by a different palindromic sequence or when the transfected cells were not superinfected, indicating the presence of latent origins of replication which could only be activated by a viral trans-acting factor(s) . Moreover, the products of resolution and replication from the two termini were characteristically different . Analysis of individual terminal fragments showed that viral 5' (right-end) sequences were resolved predominantly into "extended" structures with covalently associated copies of the virally encoded NS-1 polypeptide, while bridges derived from the 3' (left) end resolved into both NS-1-associated extended termini and lower-molecular-weight "turn-around" forms in which the two DNA strands were covalently continuous . This pattern of resolution exactly coincides with that seen at the two termini of replicative-form intermediates in normal virus infections . These results demonstrate that the bridge structures are authentic substrates for resolution and indicate that the frequency with which extended versus turn-around forms of each terminus are generated is an intrinsic property of the telomere.

Yao Xue Xue Bao, 1992, 27(1), 5 - 9
{The effect of Astragalus polysaccharide on endotoxin-induced toxicity in mice}; Wang LX et al.; Astragalus Polysaccharide (APS), an active component, was isolated from the radix of Astragalus membranaceus Bge var . mongholicus (Bge) Hsiao, and the effects of APS on E . coli endotoxin-induced liver damage were investigated in mice . The results showed that when mice were pretreated with APS (30, 60, 100 mg.kg-1.d-1 x 7d, ip), the survival rate of endotoxin intoxicated mice was increased the lowering of ATP levels and adenylate energy charge in mouse liver could be prevented . APS 100 mg.kg-1 could protect the mice from death on endotoxin (25 mg.kg-1) intoxication; and the level of ATP, the value of adenylate energy charge in the protected mouse liver were almost recovered to normal range . The effects of APS were shown to be dose-dependent . Concomitantly, the increase of MDA and decrease of GSH in mouse liver could be corrected by APS pretreatment . The results revealed that APS has an antioxidative action . An ultramicroscopic examination showed that the injury of the bio-membrane and the crest of mitochondria were ameliorated by APS pretreatment . These findings suggest that the protective effects of APS on E . coli endotoxin intoxicated mice may be due to its antioxidative action to protect the mitochondria bio-membrane, therefore, the adenylate metabolism is improved in mouse liver.

Plant Cell, 1992 Jan, 4(1), 87 - 98
DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli; Klimczak LJ et al.; To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli . Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli . The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized . It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation . The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II . Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex . The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors . The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.

Yi Chuan Xue Bao, 1992, 19(2), 177 - 85
{Expression and distribution of catechol 2,3-dioxygenase in Escherichia coli}; Xia D et al.; A series of new plasmids containing xylE gene was constructed based on the shuttle plasmid pTG 402 between E . coli and B . subtilis . The expressed xylE gene product catechol 2,3-dioxygenase (CatO2ase) was measured for its output and distribution, and analysed for its structural hydrophobicity and hydrophilicity . It was demonstrated that the output of CatO2ase was relative to plasmids, host cells, culture time and with or without induction . The enzyme didn't have features of excretional proteins and was mostly distributed inside the cells though a little could be detected in culture medium . It can be used as a selective marker, indicator and monitor in the study of genetic engineering . This research also provides a scientific basis for eliminating pollution of aromatic hydrocabonic compounds with genetic engineering bacteria.

Yi Chuan Xue Bao, 1992, 19(2), 156 - 61
{Sequence and primary structure analysis of specific DNA fragment related to pollen fertility in radish chloroplast genome}; Zhang L et al.; Nucleotide sequence of a radish chloroplast BafHI-EcoRI fragment (ZL1) derived from B21, which is related to cytoplasmic male sterility and also found in radish 401B retaining line, has been determined . The results show that this fragment is 474bp in length, and is rich in AT base components . The nucleotide sequence of ZL1 has 96.6% homology as compared with the corresponding sequence of tobacco which is located at the positions from 142330 to 142803 in IRA and from 100199 to 99726 in IRB in the tobacco chloroplast genome . It seems that ZL1 contains part of rps7-rps12 operon and encodes 7 of amino acid residues of C-terminal of rps12 as well as 93 residues of N-terminal of rps7 . The secondary structure of the partial intron from 1 to 119 in ZL1 was deduced by computer . This three stemloop structure is very similar to that of maize reported by Giese, and it has the CONSENSUS sequence of the Group II intron . The deduced amino acid sequence of S7 in ZL1 shows 95.7, 79.6, 69.9, 40.9, 36.6 and 46.2% and that of S12 shows more than 70% homology compared with the corresponding sequences of tobacco, maize, liverwort, E . gracilis, E . coli and cyanobacterium respectively . The results imply that chloroplast ribosomal proteins may play a role in cytoplasmic male sterility in higher plants.

Bioorg Khim, 1992 Jan, 18(1), 78 - 84
{Design of a DNA-matrix with a specific structure for synthesizing RNA using the polymerase chain reaction}; Smelkova NV et al.; Combination of chemical DNA synthesis and polymerase chain reactions has been used for obtaining DNA templates of preset structure coding for target mRNAs . The obtained DNA were 136 to 246 bp in length, contained promoters for RNA polymerases of phage T7 or E . coli and coded for 136-membered mRNA.

Bioorg Khim, 1992 Jan, 18(1), 63 - 70
{Cloning and expression of the gene for Escherichia coli thermostable enterotoxin}; Mikul'skis AV et al.; Gene estA coding for thermostable enterotoxin of Escherichia coli has been cloned . It is shown that in the E . coli strain SA162 this gene is located on the chromosome . Using polymerase chain reaction a site-directed mutagenesis of the cloned gene has been carried out, resulted in a recombinant strain--producer of the thermostable enterotoxin.

Bioorg Khim, 1992 Jan, 18(1), 126 - 41
{Fragments of biopolymers containing glycosylphosphates residues . 9 . Synthesis of tetra(2-acetamido-2-deoxy-D-glucopyranosyl)triphosphate-- a tetrameric fragment of the capsule antigen from Escherichia coli K51}; Nikolaev AV et al.; Linear tetra(N-acetylglucosaminyl)triphosphate GlcNAc(alpha)-P-3GlcNAc (alpha)-P-3GlcNAc(alpha)-P-3GlcNAc(beta)-ONp, a fragment of the capsular antigen of E . coli K51, was synthesized by the step-by-step approach with the use of the H-phosphonate method, starting the chain from p-nitrophenyl 2-acetamido-4,6-di-O-benzoyl-2-deoxy-beta-D-glucopyranoside . The elongation cycle included the coupling of 2-acetamido-4,6-di-O-benzoyl-2-deoxy-3-O-p-methoxybenzyl-alpha-D-gluc opy ranosyl H-phosphonate with a hydroxyl component in the presence of Me3CCOCl followed by oxidation (I2) and de (methoxybenzylation) (Ce(NH4)2(NO3)6) . 2-Acetamido-3,4,6-tri-O-benzoyl-2-deoxy-alpha-D-glucopyranosyl H-phosphonate was employed in the final step . After mild debenzoylation the title tetramer was isolated by anion-exchange chromatography . The data of 1H, 13C and 31P NMR spectra of the synthesized oligomers are discussed.

Graefes Arch Clin Exp Ophthalmol, 1992, 230(5), 463 - 7
Cellular response to intravitreal injection of endotoxin and xanthine oxidase in rabbits; McGahan MC et al.; In the present study, the ocular inflammatory response to intravitreally injected endotoxin and xanthine oxidase was studied and the cellular response of the anterior and posterior segments was contrasted . There was a clear dose response relationship to both compounds in aqueous humor protein concentration and aqueous and vitreous humor white cell number . Xanthine oxidase and low doses of endotoxin (0.25 and 1.0 ng) produce a mainly mononuclear response in the anterior segment . Higher doses of endotoxin (10 and 100 ng) produced a predominantly neutrophilic response . Cellular infiltration into the posterior segment differed qualitatively and quantitatively from the anterior segment in response to the same stimuli . Myeloperoxidase (MPO) activity (a marker for neutrophils) of the iris-ciliary body was increased only in those eyes with a large neutrophilic response and thus is not recommended for use as a definitive index of the ocular inflammatory response, but may be a useful adjunct for such studies.

C R Acad Sci III, 1992, 314(12), 527 - 32
{Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli}; Bregegere F et al.; We have fused the variable domains of a mouse antibody to the C-terminal end of the maltose-binding protein (malE), at the genetic level . The hybrid proteins were expressed in E . coli under control of the malEp promoter, and exported to the periplasm, at low temperature . They were purified by affinity chromatography on cross-linked amylose . When the two variable domains were fused together through a peptide link, the hybrid displayed similar affinity and specificity to the antigen as the native antibody.

Free Radic Res Commun, 1992, 16(1), 41 - 9
The role of extracellular medium components and specific amino acids in the cytotoxic response of Escherichia coli and Chinese hamster ovary cells to hydrogen peroxide; Brandi G et al.; A concentration of H2O2 resulting in mode one killing of Escherichia coli is more toxic when exposure to the oxidant is performed in complete medium (K medium), as compared to a saline (M9 salts) . Inorganic salts (MgSO4 and CaCl2), thiamine or glucose, when added separately, or combined, to M9 salts had no effect on the cytotoxic response to H2O2 . In contrast, the lethality of the oxidant was highly dependent on the presence of the amino acids in the incubation medium . The addition of glucose further enhanced this response . Among the seventeen amino acids which are present in the complete amino acid mixture, only two, i.e . L-histidine and L-cystine, were found to increase the toxicity of H2O2 . Again, glucose augmented this response . The effect of these amino acids on the growth inhibitory action of hydrogen peroxide was also tested in Chinese Hamster Ovary cells . It was found that L-histidine was capable of increasing the toxicity of the oxidant whereas all the other amino acids did not affect the toxicity of the oxidant . Glucose only slightly augmented this effect of L-histidine . DNA single strand breakage produced by H2O2 was increased by L-histidine and was not significantly modified by the other amino acids . DNA double strand breakage was also shown to occur in cells exposed to H2O2-L-histidine, and this effect was independent on the presence of glucose . These results demonstrate that the cytotoxic response of bacterial and mammalian cells to challenge with H2O2 is highly dependent on the composition of the extracellular milieu.(ABSTRACT TRUNCATED AT 250 WORDS)

Adv Exp Med Biol, 1992, 312, 83 - 8
Molecular characterization of HIV-2 (ROD) protease following PCR cloning from virus infected H9 cells; Cheng YS et al.; A 450 nucleotide sequence corresponding to the nucleotides 1931-2380 of the viral genome (8) was amplified by polymerase chain reaction (PCR) using template DNA prepared from HIV-2 (ROD) infected H9 cells . The sequence codes for HIV-2 protease and its N-terminal flanking peptide . An identical DNA sequence was obtained from three independent PCR amplifications, which differs from the published sequence of HIV-2 (ROD) in 7 nucleotides scattered throughout the region of the cloned DNA . The cloned DNA was expressed in E . coli cells and resulted in the synthesis of a correctly processed HIV-2 protease, which is enzymatically active . Therefore, none of the seven nucleotide changes, which resulted in two amino acid substitutions, affect the autoproteolytic or trans-cleaving activities of the HIV-2 protease.

J Basic Microbiol, 1992, 32(3), 201 - 7
A correlation with type of sulfonamide resistance gene in Escherichia coli and synergy between trimethoprim and sulfamethoxazole; Valentine CR et al.; Plasmids carrying type I or II sulfonamide-resistance (Sur) genes were evaluated for their effect on synergy between trimethoprim (Tmp) and sulfamethoxyzole (Smx) in E . coli . Strain J53 containing each of three plasmids (R1, pSa, and R388) with the type I Sur gene displayed a synergistic response to Tmp/Smx; strain LE392 containing a plasmid (RSF1010) with the type II Sur gene displayed no synergy . The difference in synergy between type I and type II Sur genes might be explained by the difference in amount of resistant enzyme produced.

Arch Microbiol, 1992, 157(5), 402 - 5
Relationship between size of parent at cell division and relative size of its progeny in Escherichia coli; Koppes LJ et al.; This article examines the empirical basis for the assumption of independence between the relative size (length or surface area) of a newborn cell w and the absolute size of its mother at cell division . Random samples from two strains of Escherichia coli B/r cells in steady-state exponential growth, covering a range of doubling times, were fixed in osmium tetroxide and prepared for electron microscopy by agar filtration . Length and diameter of over 3000 constricted cells were measured from the electron micrographs and cell surface area computed by assuming an idealized geometry of right circular cylinders with hemispherical polar caps . In general, these strains were found to divide into two daughter cells with a precision that is independent of the size of the mother . In addition, both a normal and a symmetrical beta-distribution were shown to fit the observed size distributions of w rather well; theoretical grounds for preferring the latter are discussed.

Arch Microbiol, 1992, 157(3), 229 - 34
Gas vesicles are strengthened by the outer-surface protein, GvpC; Hayes PK et al.; The critical collapse pressure of gas vesicles isolated from Anabaena flos-aquae decreased from 0.557 to 0.190 MPa when GvpC, the hydrophilic 22 kDa protein present on the outer surface of the gas vesicle, was removed by rising in 6 M urea . Recombinant GvpC was purified from inclusion bodies, produced in an E . coli strain containing an expression vector bearing the gene encoding GvpC from A . flos-aquae, and then solubilised in 6 M urea . This recombinant GvpC became bound to gas vesicles that had been stripped of their native protein, when the urea was removed by dialysis; the amount which bound increased with the concentration of GvpC present . The critical pressure of these reconstituted gas vesicles increased to 0.533 MPa, 96% of the original value . These results indicate that the function of GvpC is to increase the strength of the structure.

Mol Biol (Mosk), 1992 Jan-Feb, 26(1), 191 - 200
{Analogs of nucleotides, modified by a sugar residue and pyrimidine base, in a DNA synthesis reaction, catalyzed by Thermus aquaticus DNA polymerase}; Savochkina LP et al.; Substrate properties of dNTP analogues in the DNA synthesis reaction catalyzed by Thermus aquaticus DNA polymerase were studied . It was shown that most of dNTP analogues which were known as terminators of DNA synthesis of E . coli DNA polymerase I were able to terminate DNA synthesis catalyzed by Thermus aquaticus DNA polymerase . An interesting feature of Thermus aquaticus DNA polymerase was the ability to utilize 3'-azido-2',3'-dideoxythymidine triphosphate as terminating substrate . Relative efficiency of tested dNTP analogues incorporation into the DNA growing chain was estimated.

Mol Biol (Mosk), 1992 Jan-Feb, 26(1), 158 - 67
{Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells}; Pugachev KV et al.; On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E . coli cells . It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues . A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained . The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated . According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.

Folia Microbiol (Praha), 1992, 37(3), 181 - 7
Transformation of Streptomyces lincolnensis protoplasts with plasmid vectors; Jandova Z et al.; A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described . Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells . Transformation of S . lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2 . The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used . Interspecific transformations showed that there is no efficient restriction system in S . lincolnensis that would limit the transfer of genetic information from S . lividans or E . coli.

Environ Mol Mutagen, 1992, 20(2), 84 - 8
Mutation spectrum of spontaneous frameshift revertants in yeast using double-strand gap repair; Plewa MJ et al.; A mutation spectrum was constructed from a series of randomly isolated spontaneous His+ revertants of the frameshift mutant his4-38 in Saccharomyces cerevisiae . For each true revertant, a 438 bp region encompassing his4-38 on chromosome III was recovered into a shuttle vector by double-strand gap repair . Of the 45 independent His+ revertants sequenced, 44 were -1 base deletions and one revertant was a +2 base insertion . The -1 deletions exhibited a bimodal distribution . Of the bases encompassing the his4-38 region from +153-181, approximately 45% were not involved in a reversion event, although a -1 frameshift within this region will result in a viable His+ revertant . Approximately 49% of -1 events occurred within runs of 3 repeated bases . At these sites the strand-slippage model for frameshift mutation is supported . However, the -1 events occurring at sites of 2 repeated bases and the low frequency (2%) of +2 base insertions suggest that the transiently misaligned template model is a significant mechanism in reversion of his4-38 . When the distribution of -1 events at repeated bases was discounted, a hotspot involving a -T at position +163 was resolved.

Environ Mol Mutagen, 1992, 20(2), 140 - 4
Structure-activity relationship of genotoxic polycyclic aromatic nitro compounds: further evidence for the importance of hydrophobicity and molecular orbital energies in genetic toxicity; Debnath AK et al.; A quantitative structure-activity relationship (QSAR) has been formulated for 15 polycyclic aromatic nitro compounds acting on E . coli PQ37 . Upon damage of DNA by these substances beta-galactosidase is induced and can be easily assayed colorimetrically, hence, this is a short-term test for mutagenicity . The QSAR (log SOSIP = 1.07 log P - 1.57 epsilon LUMO - 6.41) is strikingly similar to that found earlier with nitroaromatics acting in the Ames test (TA100) and differs significantly for that found using TA98 organisms . The QSAR brings out in a unique manner the underlying similarity in the two test systems.

Mol Carcinog, 1992, 6(1), 32 - 42
Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells; Klinedinst DK et al.; We used a shuttle vector based on the Epstein-Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells . Plasmid DNA was incubated at pH 2 at 40 degrees C for various times to induce up to 20 apurinic (AP) sites per 9.7-kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient . After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid-encoded herpes simplex virus type 1-thymidine kinase gene . Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells . The mutant frequency at 20 AP sites/plasmid was 10-fold to 13-fold greater than that observed for untreated DNA . Deletion and frameshift events accounted for 46-55% of the mutants induced by depurination . The induced deletions were relatively small (median size, 100-150 bp) and characterized by short (1-5 bp) regions of sequence homology at the endpoints . These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP-site-induced template dislocation during DNA synthesis . A broad spectrum of base-substitution mutations, which accounted for 19-36% of the induced mutants, was observed . The apparent preference for insertion opposite AP sites in human cells was G (43-55%) greater than A approximately C (18-21%) greater than T (9-14%) . Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.

Eur Surg Res, 1992, 24(3), 143 - 54
Effect of recurrent endotoxemia on hemodynamics, lung function and neutrophil activation in sheep; Seekamp A et al.; The aim of the study was to find out in which way lung permeability and polymorphonuclear leukocyte (PMNL) functions are modulated under recurrent endotoxin challenge, as it might occur in clinical septic patients . In a sheep model with chronic lung lymph fistula, performing bronchoalveolar lavage (BAL), we investigated the relationship between PMNL function and endothelial as well as epithelial damage in the lung in a sepsis syndrome, using a protocol of recurrent endotoxemia induced by 1 microgram/kg body weight Escherichia coli endotoxin treatment every 12 h over a 5-day period . Pulmonary response showed constantly increased pulmonary arterial pressure at mean values of 24-30 mm Hg . Also, lymph flow did not return to baseline, but remained on a level of 6-9 ml/30 min, after an increase to 12-15 ml/30 min following each endotoxin injection . In contrast, a lower increase in protein clearance was noted upon subsequent endotoxin administration . After initial values of 7-8 ml/30 min following the first endotoxin injection, almost baseline values were measured on the 5th day (3-4 ml/30 min) . In systemic hemodynamics, we noted a decrease in cardiac output to 3.0 l/min after the first endotoxin injection, followed by a significant increase to 7 l/min under subsequent endotoxin administration . In PMNL function, we observed an attenuation of the acute response of the decrease in PMNL count, in vitro chemiluminescence response and plasma beta-N-acetylglucosaminidase level . The plasma urea concentration revealed a transient reduction in kidney function . In the epithelial lining fluid (ELF) of the alveoli, total cell count did not change significantly, but the fraction of PMNL increased from 2 to 20% during the 5 days . The ELF/plasma ratios of albumin and total protein did not change significantly . In conclusion, recurrent endotoxemia in a sheep model can produce a hyperdynamic state like in a sepsis syndrome which is further characterized by an initial leakage of the endothelial barrier, only minor affection of the epithelial barrier and by an exhaustion of PMNL function.

Curr Top Cell Regul, 1992, 33, 331 - 42
Orotidylate decarboxylase of yeast and man; Jones ME; The mechanism for ODCase appears to involve the formation of a zwitterion of OMP and a ylid on decarboxylation . Thiamin pyrophosphate catalyzes various decarboxylation and transfer reactions involving ketone groups because the thiazolium ring with its positively charged N atom can, on the loss of a proton from the adjacent C-2, generate a ylid which adds to carbonyl groups to produce a substrate ylid . The unusual aspect, then, of the ODCase reaction is that the substrate itself becomes the ylid, presumably by gaining a proton from ODCase, which results in a positive charge on the N-1 atom of the pyrimidine ring . It is a zwitterion in the transition state which momentarily becomes a ylid on decarboxylation of OMP which then yields the product, UMP . There is no known cofactor for the ODCase reaction . It will be of interest to discover the groups on the enzyme that aid in formation of the zwitterion and the ylid . Further work on the crystal structure and on the production of altered enzymes (where specific amino acids suspected to be important for the reaction are changed) should reveal more details about this important and novel reaction.

Microbios, 1992, 72(292-293), 175 - 82
Involvement of cellular division cycle in the susceptibility of Escherichia coli to cold- and osmotic-shock; Hodgson A et al.; Susceptibility of Escherichia coli to cold-shock, osmotic-shock and cold-osmotic-shock has been evaluated throughout the cellular division cycle for a variety of generation time . Two periods of sensitisation were detected . The first of these was towards cold-shock which occurred 60 min prior to cell division and might correspond to initiation of DNA replication . The second period of sensitisation was towards both cold-, osmotic- and cold-osmotic-shock and occurred immediately prior to and during cell division . Patterns and extents of sensitisation towards each of these three stresses during the second period were suggestive of a common sensitising event associated with constriction of the cell and separation.

J Basic Microbiol, 1992, 32(6), 373 - 80
Neighbour restoration: a possible explanation for some "broken" survival curves of UV-irradiated Escherichia coli K12 cells; Bernardo-Filho M et al.; Survival curves for UV irradiation of five non-filamenting strains of E . coli K12 decrease exponentially at moderate levels of radiation but are broken at high levels of radiation . That is, it appears a change in the slope of survival curves, featuring a tail . As this phenomenon was observed with strains bearing uvrA and recA mutations it must be independent of the products of these genes . Experiments with isolated surviving colonies and synchronized cultures eliminated genetic and phenotypic heterogeneity as reasons for the tails in the survival curves . UV survival increased 10- to 15-fold when UV-irradiated cells were plated either with cells unable to grow in the plating medium or with bacterial cell-free extracts . We suggest that factors related to the high cell densities used to obtain survival curves at high radiation levels (neighbour restoration) could be responsible for the survival increases, generating the tailed survival curves.

Acta Biochim Pol, 1992, 39(3), 265 - 9
Is the tRNA ochre suppressor supX derived from gltT?
Plachta A, Janion C.
Some of the argE3-->Arg+ revertants show supX suppressor activity . The genetic relationship of supX is not yet known but the evidences are presented, that supX does not derive from gltT encoding tRNA(Glu)UUG.

Arch Microbiol, 1992, 158(6), 444 - 51
The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli; Jacobi A et al.; The hyp operon of Escherichia coli comprises several genes which are required for the synthesis of all three hydrogenase isoenzymes . Deletions were introduced into each of the hypA-E genes, transferred to the chromosome and the resulting mutants were analysed for hydrogenase 1, 2 and 3 activity . The products of three of the genes, hypB, hypD and hypE were found to be essential for the synthesis of all three hydrogenase isoenzymes . A defect in hypB, as previously observed, could be complemented by high nickel concentrations in the medium, whereas the effects of mutants in the other genes could not . Lesions in hypA prevented development of hydrogenase 3 activity, did not influence the level of hydrogenase 1 but led to a considerable increase in hydrogenase 2 activity although the amount of hydrogenase 2 protein was not drastically altered . Lesions in hypC, on the other hand, led to a reduction of hydrogenase 1 activity and abolished hydrogenase 3 activity . HYPA and HYPC, besides being required for hydrogenase 3 formation, therefore may have a function in modulating the activities of the three isoenzymes with respect to each other and adjusting their levels to the requirement imposed by the physiological situation . Mutations in all five hyp genes prevented the apparent processing of the large subunits of all three hydrogenase isoenzymes . It is concluded that the products of the hypA-E genes play a role in nickel incorporation into hydrogenase apoprotein and/or processing of the constituent subunits of this enzyme . The importance of their roles is also reflected in their phylogenetic conservation in distantly related organisms.

Matrix Suppl, 1992, 1, 231 - 6
The latency of the human fibroblast collagenase precursor depends on an internal cysteine residue; Engler JA et al.; The proenzyme form of human fibroblast collagenase has been expressed in E . coli from its cDNA clone and has been shown to be functionally identical to the human enzyme . Mutants at one of three cysteine residues were constructed by site-directed mutagenesis of the cDNA and their relative activities compared to the wild type enzyme . A cysteine contained in the propeptide domain of procollagenase and other matrix metalloproteinases was shown to be essential for maintaining the proenzyme in an inactive state . A model to explain the importance of this highly conserved cysteine to the maintenance of latency is discussed.

Microbiol Immunol, 1992, 36(8), 823 - 31
A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1; Tanaka N et al.; A simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described . The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid . A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting . In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C . When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease . Recombinant p24 was purified to near homogeneity by a simple three-step procedure . The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E . coli by the coexpressed protease . The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.

Life Sci, 1992, 51(26), 2041 - 8
Cloricromene antagonizes antidipsogenic effects induced by endotoxin, but not by TNF alpha, in the rat; Calapai G et al.; Intravenous (640 micrograms/kg) or intracerebroventricular (0.5 and 1 microgram) injection of Escherichia coli endotoxin (LPS) causes inhibition of water intake induced by 24 hour period of water deprivation in the rat . Tumor necrosis factor alpha (TNF-alpha; 20 and 40 ng/rat) given into the lateral cerebral ventricle (i.c.v.) causes effects similar to those observed after LPS . Cloricromene, given either intravenously (1 and 2 mg/kg) or i.c.v . (250 and 500 ng), abolished the antidipsogenic effect induced by LPS (administered both i.v . and i.c.v.) . Cloricromene (2 mg/kg, i.v . or 500 ng/rat, i.c.v.), on the contrary, did not modify the antidipsogenic effects induced by TNF-alpha . These data indicate that peripherally injected cloricromene (as well as that i.c.v . injected) antagonizes the effects of mediators of LPS on sites regulating thirst and suggest that cloricromene's action may be due to inhibition of brain TNF-alpha formation induced by LPS.

Receptor, 1992 Summer, 2(2), 93 - 107
Polyclonal antibodies from rabbits and chickens against the estrogen receptor and related peptides . Use in the affinity isolation of estrogen receptors and the retrieval of chromatin fragments associating with estrogen receptors; Schuh TJ et al.; Polyclonal antibodies from chickens and rabbits have been prepared against polypeptides representing two regions of the human estrogen receptor (hER) . The estrogen receptor (ER) peptides used as antigens were overproduced in Escherichia coli . When indicated, the antibodies were affinity purified using resins to which the antigens contained in bacterial inclusion bodies had been coupled in high yield to epoxy-activated agarose . The antibodies recognize denatured human, bovine, rat, and rabbit ER in immunoblotting experiments . Immuno-precipitation of native ER protein was readily accomplished using rabbit antisera and immobilized protein A . The chicken antibodies, available in larger quantities, were also useful for immunoisolation after coupling to agarose . With the use of these reagents, the selective retrieval of chromatin fragments from MCF-7 cells that interact with ER has been achieved.

Receptor, 1992 Summer, 2(2), 77 - 92
Overproduction of full-length and truncated human estrogen receptors in Escherichia coli; Ahrens H et al.; The full-length human estrogen receptor (hER) as well as two overlapping peptides of hER were overproduced in Escherichia coli JM109 cells, using the inducible pIC vector system . The N-terminal receptor peptide contains the DNA-binding domain as well as the hinge region, whereas the C-terminal peptide contains the same hinge region and the hormone-binding domain . Typically, 1-6 mg of estrogen receptor (ER) peptides can be recovered from 1 L E . coli cell cultures . The majority of the overexpressed proteins are found in inclusion bodies, which allow the isolation of ER peptides in high yields and of 50-80% purity . Induction for short time periods at 10 microM inducer yielded up to 50% of the ER peptides in soluble form with full biological activity . Both the intact receptor and the C-terminal fragment specifically bound estrogens and antiestrogens, whereas ER peptides that contained the DNA-binding domain were retained on a DNA-agarose resin.

DNA Seq, 1992, 3(3), 153 - 65
The replication origin of the Methylomonas clara plasmid pBE-2; Kues U et al.; The Methylomonas clara narrow host range plasmids pBE-2 and pBE-3 belong to the class of plasmids encoding a trans acting replication initiation factor . Characteristically for such plasmids, the sequence of the origin of pBE-2 and pBE-3 contains a number of large direct repeats (8 and a half iterons of 19 bp), which by analogy are putative binding sites of the trans acting replication factor . Several additional features typical for the majority of E . coli plasmids were found in the M . clara origin: These include sequences homologous to the E . coli DnaA-box, sequences resembling E . coli IHF binding-sites, an AT-rich region with short repeats (similar to those repeats of E . coli origins responsible for an initial DNA duplex opening), and an AT-rich bent DNA region containing inverted repeats which have homology to small repeated sequences found in several plasmid origins . In addition, in the M . clara plasmid origin, large potential hairpin structures are present and the sequence of one of these participates in site specific recombination.

Vaccine, 1992, 10(13), 920 - 7
Strategies for the development of an antimalarial vaccine; Enders B et al.; Susceptible Aotus monkeys were immunized with Escherichia coli-derived fusion proteins containing partial sequences of the proteins MSAI, SERP, HRPII and with a group of three recombinant antigens isolated by screening with an antiserum raised against the protective 41 kDa protein band . HRPII, the combination of the fusion proteins of the 41 kDa group and a mixture of two sequences of SERP conferred significant protection against a challenge infection with Plasmodium falciparum blood stages . Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E . coli containing selected partial sequences . In two independent immunization trials it was shown that immunization of Aotus monkeys with either of the two hybrid proteins can protect the animals from an experimental P . falciparum infection.

Biol Neonate, 1992, 62(5), 325 - 36
Effects of injectable beta-carotene and vitamin A on lymphocyte proliferation and polymorphonuclear neutrophil function in piglets; Hoskinson CD et al.; Newborn piglets were injected with either (1) vehicle; (2) 20 mg beta-carotene; (3) 40 mg beta-carotene; (4) 25,000 IU vitamin A palmitate, or (5) 50,000 IU vitamin A palmitate . Blood was collected at 0.5, 1, 3, and 6 weeks of age and lymphocyte proliferation, polymorphonuclear neutrophil (PMN) phagocytosis and intracellular killing of live Escherichia coli were measured . Lymphocyte proliferation in all piglets was high at birth but decreased thereafter . Piglets injected with either high beta-carotene or vitamin A palmitate showed higher phytohemagglutinin- and pokeweed mitogen-induced lymphocyte proliferation at week 3 compared to controls . Similar results were seen with 20 micrograms/ml concanavalin A in piglets injected with high vitamin A palmitate . Phagocytosis by PMN isolated from all piglets decreased to a nadir at week 3 but returned to values observed in the newborn by week 6 . Treatment had no effect on phagocytosis . Piglets injected with beta-carotene or vitamin A palmitate had lower intracellular killing ability at week 3 compared to controls . Therefore, beta-carotene and vitamin A palmitate enhanced mitogen-induced lymphocyte proliferation but reduced killing ability by PMN in piglets.

Glia, 1992, 6(4), 289 - 300
Clonal segregation of oligodendrocytes and astrocytes during in vitro differentiation of glial progenitor cells; Lubetzki C et al.; To study the clonal lineage of the glial progenitor population, isolated from newborn rat brain (Lubetzki et al . J Neurochem 56:671, 1991), we combined somatic transgenesis using a retroviral vector encoding a modified bacterial beta-galactosidase with nuclear localization, and triple immunofluorescence labeling with A2B5, anti-galactosylceramide, and anti-glial acidic fibrillary protein antibodies . This allowed clonal analysis of the postnatal glial lineage with precise phenotypic identification of each cell within the lacZ-positive clones . When infected cells were cultivated under constant conditions, in the presence of either 1% or 10% fetal calf serum (FCS)-containing medium, all the 250 lacZ-positive clusters examined were homogeneous, i.e., either oligodendroglial or astroglial . Mixed astrocyte-oligodendroglial clones were observed when cells cultivated in the presence of 1% FCS were switched to a 10% FCS-containing medium, confirming the bipotentiality of glial progenitor cells (Temple and Raff Nature 313:223, 1985) . However, even under the switch culture conditions, segregation into homogeneous clones of either oligodendrocytes or astrocytes still predominated, and the percentage of mixed clones dropped from 25 to 8 or to 3, when the switch took place at 8, 16, or 22 days in vitro, respectively . Two additional observations lead us to suggest that microenvironmental factors are responsible for the clonal segregation of glial progenitor cells: 1) the uneven distribution of oligodendrocyte and astrocyte clusters, the latter being seen mostly on the edge of the coverslips; and 2) the presence, in the vicinity of an homogeneous lacZ-positive clone, of some lacZ-negative cells expressing the same phenotype.

Agents Actions Suppl, 1992, 38 ( Pt 3), 413 - 20
CP-0127, a novel potent bradykinin antagonist, increases survival in rat and rabbit models of endotoxin shock; Whalley ET et al.; The bradykinin antagonist dimer CP-0127 was found to be a potent and selective inhibitor of the depressor response to bradykinin in the anaesthetized rat and rabbit . When given as a single dose s.c . (3.6 mumol/kg), the depressor response to bradykinin was blocked for the duration of the experiment (4 hours) . In anaesthetized control rats, LPS from E . coli produced a profound and immediate hypotensive response, while in rats infused with CP-0127, the response to LPS was almost totally reversed . In addition, CP-0127 given as a single subcutaneous dose (3.6 mumol/kg) to rats 1 hour before LPS challenge produced a 93% survival rate, compared to 14% in control animals . Finally, a survival rate of 86% was achieved in rabbits infused with CP-0127 at 0.36 nmol/kg/min i.v., compared to 45.5% in saline-infused control animals given LPS (500 micrograms/kg i.v.) . The results of these experiments provide evidence for a significant role for the kallikrein-kinin system in these models of endotoxic shock, and indicate the therapeutic potential of a bradykinin antagonist such as CP-0127 for treating this disorder in man.

Agents Actions Suppl, 1992, 38 ( Pt 3), 376 - 84
Plasma exudation by activated plasma kallikrein after intraperitoneal injection of lambda-carrageenin or endotoxin in rats; Sugimoto K et al.; The experimental study was carried out to examine whether intraperitoneal injection of carrageenin or endotoxin activates plasma kallikrein-kinin system to induce plasma exudation in rats . Intraperitoneal injection of 2% lambda-carrageenin induced plasma exudation in the peritoneal cavity by activation of plasma prekallikrein . Intraperitoneal injection of endotoxin (3mg/kg) also resulted in intraperitoneal plasma exudation, but plasma kallikrein-kinin system did not seem to be involved.

Roum Arch Microbiol Immunol, 1992 Jan-Jun, 51(1-2), 5 - 16
Development of an irradiated vaccine that protects against enterotoxigenic Escherichia coli diarrhoea; Dima VF et al.; In the pathogenesis of diarrhoea in man bacteria adhesion to enterocytes is mediated by specific CFA/I or CFA/II antigens . A perorally administered vaccine was prepared from E . coli H10407 (078:H11) by irradiation with electrons with high energy (EHE) . Two hours after cimetidine administration rats were immunized per os with 5 irradiated vaccine doses at 4-day intervals . Seven days after the last immunization animals were infected by inoculating 1 x 10(9) germs in the ligated intestinal loop . Reduction of the intestinal secretion by over 50% 18 hours after inoculation was considered an efficient protection marker . The obtained results have proved a significant reduction of the intestinal secretion in immunized animals infected with serotypes 078:H11(63 +/- 4%) and 078:H12(59 +/- 5%) as compared to non-immunized animals . Experimental induction of the intestinal protection against Escherichia coli enterotoxigenic (ETEC) strains points to the possibility of using this type of irradiated vaccine in the prophylaxis of diarrhoea in man.

Chin J Biotechnol, 1992, 8(1), 23 - 8
Significance of the 56th tyrosine on cytotoxic activity of tumor necrosis factor; Zhang D et al.; After using site-specific mutagenesis a conserved amino acid residue, the 56th tyrosine in human tumor necrosis was substituted by glutamine . The expression level of the mutant protein in E . coli did not changed significantly, however, its specific activity of the cytotoxicity on L929 cultural cells was decreased to 1/583 of that of the unmutant tumor necrosis factor . The results show that the 56th tyrosine residue or the region where tyrosine 56th located is important for the cytotoxic activity of tumor necrosis factor.

Chin J Biotechnol, 1992, 8(1), 1 - 13
Cloning and expression of midecamycin 4"-acylase gene in spiramycin producing strains; Wang Y et al.; A recombinant plasmid p66B containing the midecamycin 4"-acylase gene was obtained by cloning this gene into plasmid vetor pIJ680 from the primary clone pCN6C5, presumably harboring the midecamycin biosynthetic gene . The expression of the midecamycin 4"-acylase gene (p66B) in spiramycin producing strains resulted mainly in the production of 4"-isovalerylspiramycin . Another positive clone pCN10F5 was discovered from the genomic library of S . mycarofacians 1748 by probing with p66B DNA BamHI-BamHI 2.3kb fragment . A BamHI-BamHI 8.0kb homologous region on pCN10F5 was determined by Southern hybridization and was subcloned into plasmids pWHM3 and pIJ680 . Recombinant plasmids pWF5 and p6F5 with molecular size about 15.2kb and 13.3kb, respectively, were obtained . Transformation of spiramycin producing strains with these plasmids resulted in the production of two major components . Based on their physicochemical properties and spectral evidences, component I was identified as 4"-propionylspiramycin III, and component II as 4"-propionylspiramycin II . Southern hybridization confirmed that the BamHI-BamHI 8.0kb fragment was cloned in the spiramycin producing strain . Only pCN10F5 clone was identified from the genomic library of S . mycarofaciens 1748 when the 4"-isovaleryltransferase gene of carbomycin producing strain S . thermotolerans was used as a probe in colony hybridization . It suggests that there is a difference between the 4"-acyltransferase genes in the pCN6C5 and pCN10F5 clones.

Arch Virol, 1992, 127(1-4), 117 - 37
Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine; Wagner R et al.; Antigens in a particulate conformation were shown to be highly immunogenic in mammals . For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal . Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively . Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only . In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus . Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles . Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system . The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells . Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene . Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus . However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation . Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization . Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

Zh Mikrobiol Epidemiol Immunobiol, 1992, (5-6), 17 - 21
{The phenomenon of tachyphylaxis in the pathogenesis of an experimental infectious process}; Barsukov VS; Experiments on mice have revealed that the development of experimental purulent infection is accompanied by a considerable increase in the nonspecific resistance of the animals to additional infection with unrelated bacteria (the effect of tachyphylaxis) . Morphologically, this is manifested by the rapid limitation of the focus of inflammation at the site of inoculation of the superinfecting agent . The state of the phagocytic apparatus is of great importance for this phenomenon as disturbances in the macrophage activity caused by the injection of carrageenan abolish the protective effect of primary infection . The phenomenon of tachyphylaxis has been shown to play a certain role in the prevention of the septic generalization of the process and in resistance to superinfection.

Scand J Clin Lab Invest Suppl, 1992, 210, 51 - 8
Protein engineering of the milk-clotting aspartic proteinases; Aikawa J et al.; Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) are members of the aspartic proteinases used as milk-coagulants in cheese industry . A system for production of recombinant chymosin as inclusion bodies in Escherichia coli cells and its refolding into the active form was established . Another expression system for production of Mucor rennin in Saccharomyces cerevisiae was also established . Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form . Glycosylation affected both the secretion and the enzyme properties . Site-directed mutagenesis of the Tyr residue at position 75 in chymosin and Mucor rennin revealed its crucial role in catalytic function of the aspartic proteinases . The results also suggested possibility to improve practical properties of the milk-clotting enzymes by site-directed mutagenesis.

Arch Virol Suppl, 1992, 4, 29 - 35
HBcAg induced T-cell independent anti-HBc production in chronic HBsAg carriers; Sylvan SP et al.; The capacity of the nucleocapsid protein of HBV to function as a T-cell independent antigen in man was studied . When T-cell depleted B-cell cultures were challenged with E coli-derived HBcAg, anti-HBc production was registered in culture supernatants from the majority of chronic HBsAg carriers in a quiescent stage of disease . In contrast, similarly prepared and stimulated cultures from donors with natural acquired immunity to hepatitis B or HB-susceptible controls were non-responsive . Addition of autologous T-cells effectively restored anti-HBc responsiveness in T-cell depleted B-cell cultures from HB-immune donors, demonstrating the T-cell dependency for anti-HBc induction in natural HBV-infection.

Acta Physiol Scand Suppl, 1992, 607, 31 - 40
An atomic model for the structure of bacteriorhodopsin, a seven-helix membrane protein; Ceska TA et al.; A three-dimensional map of bacteriorhodopsin has been obtained, at near-atomic resolution, by collecting and analysing electron diffraction patterns and electron micrographs from crystals of bacteriorhodopsin preserved at very low temperatures . The map shows a resolution of 3.5 degrees in a direction parallel to the plane of the membrane, but poorer resolution perpendicular . It shows many features well resolved from the main density of the seven alpha-helices, which we interpret as the bulky sidechains of tyrosine, phenylalanine and tryptophan, as well as a very dense feature, which is the beta-ionone ring of the retinal chromophore . Using these bulky side chains as starting points and taking account of bulges of density for the smaller side chains such as leucine, we built an atomic model for the residues between 8 and 225 . There are 21 amino acids from all 7 helices surrounding the retinal and 26 amino acids contributed by 5 helices that form the proton channel . Ten of the amino acids in the middle of the proton channel are also part of the retinal-binding site . The model provides a useful basis for considering the mechanism of proton pumping and in the interpretation of other experimental data . In particular, the model suggests that the pK changes in the Schiff base must act as the means by which light energy is converted to proton pumping through the channel . Asp-96 is on the pathway from the cytoplasm to the Schiff base and asp-85 on the pathway from the Schiff base to the extracellular surface . The experimental map and the building of the model of the structure will be described, as well as our interpretation of the structural basis of the mechanism.

Acta Physiol Scand Suppl, 1992, 607, 275 - 8
Expression of subunit II of chloroplast H(+)-ATPase in an Escherichia coli mutant lacking subunit b of its H(+)-ATPase; Schmidt G; The DNA of subunit II of the H(+)-ATPase from spinach chloroplast was expressed in Escherichia coli . It was found that a high gene dose is lethal to E . coli . With a lower gene dose subunit II was not able to substitute for the homologous subunit b in the E . coli ATP synthase.

Acta Physiol Scand Suppl, 1992, 607, 265 - 8
A novel type haem group of cytochrome o from Escherichia coli; Puustinen A; The b-type haem groups of cytochrome o have generally been thought to be protohaems . However, we have recently shown that they are of a novel kind, for which we propose the name haem O . On the basis of its pyridine haemochrome spectrum, chromatographic behaviour and molecular mass of 839 Da, we suggest that haem O is a haem A-like molecule with the formyl group in position 8 of the latter replaced by methyl, but with retention of the 17-carbon hydroxyethyl-farnesyl side chain in position 2, characteristic of haem A . This structure has now been unambiguously verified by resonance Raman, 1H-NMR, and infrared spectroscopy, as well as by mass spectroscopy of molecular fragments, in collaboration with C . K . Chang, G . T . Babcock and their co-workers at Michigan State University . Properties of haem O are compared with haem A and protohaem, and possible contributions of haem O structure to the cytochrome o oxidase function are discussed.

Arch Microbiol, 1992, 158(1), 68 - 73
An Escherichia coli mutant containing only demethylmenaquinone, but no menaquinone: effects on fumarate, dimethylsulfoxide, trimethylamine N-oxide and nitrate respiration; Wissenbach U et al.; The mutant strain AN70 (ubiE) of Escherichia coli which is known to lack ubiquinone (Young IG et al . 1971), was analyzed for menaquinone (MK) and demethylmenaquinone (DMK) contents . In contrast to the wild-type, strain AN70 contained only DMK, but no MK . The mutant strain was able to grow with fumarate, trimethylamine N-oxide (TMAO) and dimethylsulfoxide (DMSO), but not with nitrate as electron acceptor . The membranes catalyzed anaerobic respiration with fumarate and TMAO at 69 and 74% of wild-type rates . DMSO respiration was reduced to 38% of wild-type activities and nitrate respiration was missing (less than or equal to 8% of wild-type), although the respective enzymes were present in wild-type rates . The results complement earlier findings which demonstrated a role for DMK only in TMAO respiration (Wissenbach et al . 1990) . It is concluded, that DMK (in addition to MK) can serve as a redox mediator in fumarate, TMAO and to some extent in DMSO respiration, but not in nitrate respiration . In strain AN70 (ubiE) the lack of ubiquinone (Q) is due to a defect in a specific methylation step of Q biosynthesis . Synthesis of MK from DMK appears to depend on the same gene (ubiE).

Radiat Environ Biophys, 1992, 31(4), 343 - 8
Induction of SOS repair by ionizing radiation . Results from experiments at accelerators; Koudela K et al.; beta-galactosidase and alkaline phosphatase activities of Escherichia coli strain PQ37 carrying the fusion gene of sulA and lacZ treated with different types of ionizing radiation were examined . The induction factor (ratio of beta-galactosidase to alkaline phosphatase activity), reflecting the SOS-induction potency, increases significantly with radiation dose . Maximum effectiveness to induce SOS-response has been found for deuterium and helium ions in comparison to gamma-rays, carbon or krypton ions . Increased energy of helium ions leads to greater SOS-induction potency of radiation.

Parasitol Res, 1992, 78(6), 529 - 33
Detection and quantitation of cell-surface sugar receptor(s) of Leishmania donovani by application of neoglycoenzymes; Schottelius J et al.; Promastigote culture forms of the log growth phase of Leishmania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme . Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety was the only variable feature of the applied tools . Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of beta-galactosidase from Escherichia coli: beta-D-lactose, beta-D-thiogalactose, alpha-D-mannose, alpha-L-rhamnose, alpha-D-N-acetylgalactosamine, beta-D-N-acetylgalactosamine, beta-D-N-acetylglucosamine, the alpha- and beta-glucosides maltose and cellobiose, beta-D-xylose, alpha-D-mannose-6-phosphate, the alpha-galactoside melibiose, alpha-L-fucose, and beta-D-glucuronic acid as well as sialic acid . Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group . This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigote Leishmania . Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, and N-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealed KD values of around 100 mM and around 10(4) binding sites for the polyvalent ligands.

Int J Cancer Suppl, 1992, 7, 45 - 50
Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells; Rodrigues ML et al.; We recently constructed a humanized bispecific antibody (BsF(ab')2v1) by separate E . coli expression of each Fab' arm followed by directed chemical coupling in vitro . BsF(ab')2 v1 (anti-CD3/anti-p185HER2) was demonstrated to retarget the cytotoxic activity of human CD3+ CTL in vitro against the human breast-tumor cell line, SK-BR-3, which over-expresses the p185HER2 product of the proto-oncogene HER2 . Our minimalistic humanization strategy is to install as few murine residues as possible into a human antibody in order to recruit antigen-binding affinity and biological properties comparable to that of the murine parent antibody . This strategy proved very successful for the anti-p185HER2 arm of BsF(ab')2 v1 . In contrast BsF(ab')2 v1 binds to T cells via its anti-CD3 arm much less efficiently than does the chimeric BsF(ab')2 which contains the variable domains of the murine parent anti-CD3 antibody . Here we have constructed additional BsF(ab')2 fragments containing variant anti-CD3 arms with selected amino acid replacements in an attempt to improve antibody binding to T cells . One such variant, BsF(ab')2 v9, was created by replacing 6 residues in the second hypervariable loop of the anti-CD3 heavy chain variable domain of BsF(ab')2 v1 with their counterparts from the murine parent anti-CD3 antibody . BsF(ab')2 v9 binds to T cells (Jurkat) much more efficiently than does BsF(ab')2 v1 and almost as efficiently as the chimeric BsF(ab')2 . This improvement in the efficiency of T-cell binding of the humanized BsF(ab')2 is an important step in its development as a potential therapeutic agent for the treatment of p185HER2 over-expressing cancers.

What Is Amino Acid?, What Is Biofilter?, What Is Cell Biology?, What Is Botulism?, What Is MIC?, n, Bacteriology, s, Bacterium, o, Microbes, c, Bacteria, n, Microorganisms, a, Haemophilus, c, Streptococci, e, Staphylococcus, o, Antibiotics, o, Gram positive, r, Anaerobic bacteria, a, Lactobacillus, o, Streptococcal, a, Bacillus anthracis, s, Meningococcus, a, Salmonella, a, Microorganism, r, Antibiotics, a, Pseudomonas aeruginosa, e, Haemophilus, c, Bacillus, a, Escherichia coli, a, Cryptococci, n, Pseudomonas, i, Escherichia coli, c, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005