Escherichia coli

Biochemistry, 1988 Jan 12, 27(1), 206 - 12
Inhibition of phosphatase and sulfatase by transition-state analogues; Stankiewicz PJ et al.; The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase . Vanadate was a potent inhibitor of all three enzymes . Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-) . The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase . Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions . Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase . Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol . It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes . The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed.

Biochemistry, 1988 Jan 12, 27(1), 142 - 50
NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation; LeMaster DM et al.; All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin . Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media . The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity . In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings . Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective alpha- and/or beta-deuteriation into a fractionally deuteriated background . Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein . In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY spectra offered superior sensitivity to that observed for natural abundance samples . The resultant sequential assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 alpha-amide, beta-amide, and amide-amide connectivities.

Biochemistry, 1988 Jan 12, 27(1), 436 - 45
Preferential location of bulged guanosine internal to a G.C tract by 1H NMR; Woodson SA et al.; A series of double-helical oligodeoxyribonucleotides of sequence corresponding to a frame-shift mutational hot spot in the lambda CI gene, 5'-dGATGGGGCAG, are compared by proton magnetic resonance spectroscopy at 500 MHz of the exchangeable protons . Duplexes containing an extra guanine in a run of two, three, and four G.C base pairs are compared to regular helices of the same sequence and to another sequence containing an isolated bulged G, 5'-dGATGGGCAG.dCTGCGCCATC . The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy . Resonances assigned to the G tract in bulge-containing duplexes are shifted anomalously upfield and are very broad . Imino proton lifetimes are determined by T1 inversion-recovery experiments . The exchange rates of G-tract imino protons in bulged duplexes are rapid compared to those in regular helices and are discussed in terms of the apparent rate of solvent exchange for the isolated G bulge . Delocalization of a bulged guanosine in homopolymeric sequences can explain the observed changes in chemical shift and relaxation times across the entire G.C run, and the chemical shifts can be fit by a simple model of fast exchange between base-paired and unpaired states for the imino protons . This allows us to calculate the relative occupancies of each bulge site . In these sequences, we find the extra base prefers positions internal to the G tract over those at the edge.

Biochemistry, 1988 Jan 12, 27(1), 481 - 6
Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones; Mutzel R et al.; lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells . Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit . The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction . The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence . The results show that the Dictyostelium R subunit can be functionally expressed in E . coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein . In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit . One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D . discoideum.

Nucleic Acids Res, 1988 Jan 11, 16(1), 61 - 76
Promoters of Mycoplasma capricolum ribosomal RNA operons: identical activities but different regulation in homologous and heterologous cells; Gafny R et al.; The 5' region of the rRNA operon, rrnA, of M . capricolum was cloned . Sequence analysis revealed two tRNA genes, tRNA(leu) and tRNA(lys), upstream to the promoter of the rRNA operon . The in vivo transcription start sites of the rRNA operon and of the tRNA genes were mapped . The same promoters used by M . capricolum RNA polymerase are also recognized by E . coli RNA polymerase both in vivo and in vitro . We find that high levels of ppGpp in E . coli, resulting from amino acid starvation or from spoT mutation, activate rather than repress the transcription of the mycoplasma rrnA operon.

Nucleic Acids Res, 1988 Jan 11, 16(1), 265 - 77
Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity; Kotewicz ML et al.; Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide . Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity . It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity . We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E . coli, and purified the protein to near homogeneity . The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity . These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains.

J Biol Chem, 1988 Jan 5, 263(1), 527 - 34
Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site; Shi YB et al.; We have probed the interaction of T7 RNA polymerase with DNA in an elongation complex arrested by a site specifically placed psoralen diadduct or furanside monoadduct using DNase I footprinting techniques . The psoralen derivative, HMT (4'-hydroxy-methyl-4,5',8-trimethylpsoralen), was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing a T7 RNA polymerase promoter at one end . The psoralen molecule was photochemically attached either to 2 adjacent thymidine residues on opposite strands as a diadduct or to only 1 thymidine residue on the coding strand as a furan-side monoadduct . Using these psoralen-modified DNAs as templates for transcription, we found that T7 RNA polymerase was blocked at the psoralen adduct site and that the arrested elongation complex protected about 15 nucleotides upstream from the adduct on the coding strand and 20 nucleotides around the adduct on the noncoding strand from DNase I digestion . The two psoralen-modified DNA templates yielded identical RNA transcripts and DNase I footprints . In contrast, T7 polymerase protected only the coding strand from -20 to +8 in the initiation complex . These results suggest that the RNA polymerase undergoes a marked conformational change upon converting from an initiation complex to an elongation complex.

J Biol Chem, 1988 Jan 5, 263(1), 404 - 8
Characterization of the internal signal-anchor domain of Escherichia coli leader peptidase; Dalbey RE et al.; Leader peptidase, an integral transmembrane protein of Escherichia coli, is synthesized without a cleavable amino-terminal leader peptide . Of the five domains that participate in the membrane assembly of this protein, one is an internal "signal" region . We have used oligonucleotide-directed mutagenesis to examine the properties of the internal signal that are crucial for leader peptidase assembly . For this purpose, the net charge at the amino terminus of the internal signal was changed from +2 to +1 and -1 and, at the carboxyl terminus of the signal, from 0 to -1 or +1 . These mutations had no effect on the membrane assembly of leader peptidase, suggesting that the charges have little role in the signal function . The apolar core of this signal was disrupted by substitution of basic amino acids for apolar residues . Substitution of an arginyl residue at position 70, or two arginyl residues at position 67 and 69, prevented membrane assembly . However, substitution of an arginyl residue at position 66 or either arginyl or lysyl residue at position 68 was without effect . Thus, while the apolar character of the internal signal is important, the precise position of a charged residue determines its effect on assembly.

J Biol Chem, 1988 Jan 5, 263(1), 344 - 9
Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing; Freudl R et al.; The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene . In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus . In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues . The pro-OmpA protein is normally processed partially co- and partially posttranslationally . Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational . Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane . The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis . Hence, it does not matter which mode of processing, or rather translocation, is used . The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor . In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues . This peptide was also rapidly removed . Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.

J Biol Chem, 1988 Jan 5, 263(1), 314 - 20
Facilitated diffusion of p-nitrophenyl-alpha-D-maltohexaoside through the outer membrane of Escherichia coli . Characterization of LamB as a specific and saturable channel for maltooligosaccharides; Freundlieb S et al.; LamB, an outer membrane protein of Escherichia coli, is a component of the maltose-maltooligosaccharide transport system . We used p-nitrophenyl-alpha-D-maltohexaoside, a chromogenic analog of maltohexaose, and a periplasmic amylase that hydrolyzes this compound to study the LamB-mediated diffusion of p-nitrophenyl-alpha-D-maltohexaoside into the periplasm . Using this approach, we were able to characterize LamB in vivo as a saturable channel for maltooligosaccharides . Permeation through LamB follows Michaelis-Menten kinetics, with a Km of 0.13 mM and a Vmax of 3.3 nmol/min/10(9) cells . Previous studies suggested that maltose-binding protein increases the rate of maltooligosaccharide diffusion through LamB . We show here that, at least in strains that are unable to transport maltooligosaccharides into the cytoplasm, maltose-binding protein does not influence the rate of substrate diffusion . The periplasmic amylase had been previously described as being of the alpha-type . We have now purified this protein and analyzed its mode of action using chromogenic maltooligosaccharides of varying length . Analysis of the hydrolytic products revealed that the enzyme recognizes its substrate from the nonreducing that the enzyme recognizes its substrate from the nonreducing end and preferentially liberates maltohexaose, in contrast to the behavior of classical alpha-amylases that are endohydrolases . Using p-nitrophenyl-alpha-D-maltohexaoside as a substrate, we determined a Km of 3 microM and a Vmax of 0.14 mumol/min/mg of protein.

J Biol Chem, 1988 Jan 5, 263(1), 243 - 6
Increased binding of operator DNA by trp superrepressor EK49; Klig LS et al.; The mechanism of superrepression by the mutant trp repressor EK49 was examined . This superrepressor has a glutamic acid-to-lysine change at residue 49 . The purified EK49 trp repressor was found to have a 10-fold higher affinity than wild type repressor for trp operator DNA . This increased affinity was shown to be due to a decrease in dissociation rate . The binding of trp operator DNA by EK49 trp repressor in the filter binding assay was more sensitive to high salt concentrations than binding by wild type repressor.

J Biol Chem, 1988 Jan 5, 263(1), 200 - 9
Reversibility of strand invasion promoted by recA protein and its inhibition by Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein; Chow SA et al.; When recA protein promotes homologous pairing and strand exchange involving circular single strands and linear duplex DNA, the protein first polymerizes on the single-stranded DNA to form a nucleoprotein filament which then binds naked duplex DNA to form nucleoprotein networks, the existence of which is independent of homology, but requires the continued presence of recA protein (Tsang, S . S., Chow, S . A., and Radding, C . M . (1985) Biochemistry 24, 3226-3232) . Further experiments revealed that within a few minutes after the beginning of homologous pairing and strand exchange, these networks began to be replaced by a distinct set of networks with inverse properties: their formation depended upon homology, but they survived removal of recA protein by a variety of treatments . Formation of this second kind of network required that homology be present specifically at the end of the linear duplex molecule from which strand exchange begins . Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein largely suppressed the formation of this second population of networks by inactivating the newly formed heteroduplex DNA, which, however, could be reactivated when recA protein was dissociated by incubation at 0 degrees C . We interpret these observations as evidence of reinitiation of strand invasion when recA protein acts in the absence of auxiliary helix-destabilizing proteins . These observations indicate that the nature of the nucleoprotein products of strand exchange determines whether pairing and strand exchange are reversible or not, and they further suggest a new explanation for the way in which E . coli single-stranded DNA-binding protein and gene 32 protein accelerate the apparent forward rate of strand exchange promoted by recA protein, namely by suppressing initiation of the reverse reaction.

J Biol Chem, 1988 Jan 5, 263(1), 472 - 9
Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator; Lynn SP et al.; The thr operon of Escherichia coli is regulated by an attenuation mechanism in which regulated transcription termination occurs in response to the levels of charged tRNAthr and tRNAile . Transcription of the thr operon regulatory region in vitro produces a 162-base transcript that is terminated efficiently at the attenuator . The attenuator sequence is similar to other rho-independent terminators . It contains a G + C region of dyad symmetry followed by a run of 9 A + T residues . We have characterized in detail the sequence requirements for efficient transcription termination in vitro . Using a set of point mutations in the G + C region of dyad symmetry of the thr attenuator, we have characterized the effects of these mutations on the efficiency of transcription termination . The efficiency was reduced in all of the mutants analyzed with the greatest effect being an approximate 20% decrease in termination . In some instances the electrophoretic mobilities of the terminated transcripts on 8% polyacrylamide, 8 M urea gels were shifted substantially relative to the wild type-terminated transcript, but the sites of transcription termination were altered by only a few base pairs . We also constructed a set of deletions removing consecutive thymidines which follow the G + C-rich region of dyad symmetry . Removal of 1 or 3 of the 9 thymidine residues had no effect on termination efficiency in vitro or in vivo . Removal of four to six thymidines caused a linear decrease in the efficiency of termination . When only one or two thymidines were present in the template, termination was completely abolished . These results indicate that both the integrity of the RNA stem and the length of the consecutive thymidine residues are important signals recognized by RNA polymerase during transcription of the thr operon regulatory region.

J Biol Chem, 1988 Jan 5, 263(1), 76 - 83
High salt activation of recA protein ATPase in the absence of DNA; Pugh BF et al.; The recA protein of Escherichia coli is a DNA-dependent ATPase . In the absence of DNA, the rate of recA protein-promoted ATP hydrolysis drops 2000-fold, exhibiting an apparent kcat of approximately 0.015 min-1 . This DNA-independent activity can be stimulated to levels approximating those observed with DNA by adding high concentrations (approximately 2M) of a wide variety of salts . The increase in ATP hydrolysis appears to require the minimal interaction of three to four ions with recA protein . The active species in ATP hydrolysis is an aggregate of recA protein . There appears to be little or no cooperativity with respect to ATP binding (Hill coefficient = 1.0) . The salt-stimulated ATP hydrolysis reaction is dependent upon Mg2+ ions and is optimal between pH 7.0 and 8.0 . In many respects, the high salt concentration appears to be functionally mimicking DNA in activating the recA protein ATPase.

J Biol Chem, 1988 Jan 5, 263(1), 436 - 42
Simian virus 40 large T antigen DNA helicase . Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements; Wiekowski M et al.; The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity . The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates . The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C . For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction . The minimum length of the single-stranded tail was determined to be less than 5 nucleotides . Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction . This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase . Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome . The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.

J Biol Chem, 1988 Jan 5, 263(1), 383 - 92
The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity; Goetz GS et al.; The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined . A variety of DNA substrates were used to characterize this ATP-dependent activity . Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction . Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility . ATP and MgCl2 were required for this reaction . With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive . The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates . In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence . This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein . When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound . This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate . The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2) . Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound . In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.

J Mol Biol, 1988 Jan 5, 199(1), 35 - 45
Effect of dam methylation on Tn5 transposition; Yin JC et al.; The effect of dam methylation on Tn5 transposition was investigated by analyses of mutations in the host (Escherichia coli) and the element . Wild-type elements transposed at a higher frequency and showed higher levels of transposase expression in a dam-host . Mutations were made in the promoter region of the transcript that codes for the transposase . Transposition and transposase levels from these mutants were independent of the host methylation system . Measurements of the amount of RNA support the hypothesis that dam methylation exerts its effect on Tn5 transposition by modulating the frequency of transcriptional initiation of the transposase gene . Since Tn5 transposition increases when the transposase levels increase, at normal concentrations the amount of transposase is a rate-limiting factor that determines the transposition frequency of Tn5 . Transposition of IS50, one of the insertion sequences that constitutes Tn5, is also sensitive to dam methylation by a second mechanism in addition to that of modulating transcriptional initiation . dam methylation, either directly or indirectly, inhibits the usage of IS50 sequences by the transposase . Thus, dam methylation can affect both the expression of the transposase and the DNA substrate upon which it acts.

J Mol Biol, 1988 Jan 5, 199(1), 137 - 47
Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli; Wagenknecht T et al.; Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing . Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens . Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens . The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA . The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.

J Mol Biol, 1988 Jan 5, 199(1), 115 - 36
A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit; Brimacombe R et al.; A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model . All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E . coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis . The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering . The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit . The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions . The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits . These two distances both involve protein S13, a protein noted for its anomalous behaviour . A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch" . Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model . In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e . internal) positions in the model . All nine of the modified bases in the E . coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit . Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E . coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1988 Jan 4, 170(3), 515 - 20
cDNAs for canavalin and concanavalin A from Canavalia gladiata seeds . Nucleotide sequence of cDNA for canavalin and RNA blot analysis of canavalin and concanavalin A mRNAs in developing seeds; Yamauchi D et al.; By a method of Escherichia coli expression-vector-primed cDNA synthesis, a cDNA expression library was constructed from total poly(A)-rich RNA that was prepared from immature embryos of Canavalia gladiata . Essentially full-length cDNA clones for two seed proteins, canavalin and concanavalin A, were selected from the library by immunological screening of the colonies and in vitro RNA synthesis and translation . The complete amino acid sequence of canavalin was determined from the nucleotide sequence of the corresponding cDNA and was found to be very homologous to 7S seed proteins of other legumes . The nucleotide sequence of the cDNA predicts a 26-amino-acid extension in the precursor at the amino terminus of the mature canavalin . Canavalin mRNA and concanavalin A mRNA levels at successive stages of the seed development were estimated by RNA blot hybridization and results indicated that the two mRNA levels are differently regulated.

FEBS Lett, 1988 Jan 4, 226(2), 291 - 6
Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes; Tanimoto T et al.; The direct binding protein(s) of ras p21 was (were) investigated in inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E . coli . The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody, ras p21 bound to vesicles . Prior digestion of the vesicles with trypsin reduced this binding significantly . When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS-polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6 . ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein . These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell-free system.

FEBS Lett, 1988 Jan 4, 226(2), 255 - 60
Does the channel for nascent peptide exist inside the ribosome? Immune electron microscopy study; Ryabova LA et al.; MS2 phage RNA-directed synthesis of an N-terminal polypeptide of the phage coat protein on Escherichia coli 70 S ribosomes was initiated in a cell-free system with the N-dinitrophenyl derivative of methionyl-tRNAFMet) and performed in the absence of tyrosine, lysine, cysteine and methionine . As a result, the translating ribosomes carried peptides up to 42 amino acid residues in length with the dinitrophenyl hapten at the N-ends . Using the immune electron microscopy technique the positions of the nascent peptide N-ends on the 70 S ribosomes have been visualized . It has been found that (i) the N-ends of nascent peptides of these lengths are accessible to antibodies, (ii) the exit site of a nascent peptide is the pocket between the base of the central protuberance and the L1 ridge on the 50 S subunit, i.e . presumably its peptidyl transferase center, and (iii) the further pathway of a nascent peptide seems to proceed along the groove on the external surface of the 50 S subunit.

Eur J Biochem, 1988 Jan 4, 170(3), 637 - 42
ATP4, the structural gene for yeast F0F1 ATPase subunit 4; Velours J et al.; A plasmid containing the gene coding for the Saccharomyces cerevisiae F0F1 ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure . The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors . A 732-base-pair open reading frame encoding a 244-amino-acid polypeptide is described . The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35-amino-acid leader sequence . Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da . This subunit presents amphiphilic behaviour with two distinct domains . A high alpha-helix content of 77% was predicted from the sequence . Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase.

Eur J Biochem, 1988 Jan 4, 170(3), 627 - 30
F0 part of the ATP synthase from Escherichia coli . Influence of subunits a, and b, on the structure of subunit c; Steffens K et al.; Four different sets of proteoliposomes were prepared from F0, subunit c, a complex of subunits a and c (ac complex) and an ac complex supplemented with subunit b . Only liposomes containing intact F0 or all subunits of F0 were active in proton translocation and F1 binding {Schneider, E . and Altendorf, K . (1985) EMBO J . 4, 515-518} . The conformation of subunit c in the different preparations was analyzed by labelling the proteoliposomes with the hydrophobic photoactivatable reagent 3-(trifluoromethyl)-3-(m-{125I}iodophenyl)diazirine ({125I}TID) . Subsequent isolation and Edman degradation of this polypeptide revealed distinct radioactive labelling patterns over the entire amino acid sequence . In the F0 complex and in the ac complex subunit c retains a labelling pattern which is related to that found in TID-labelled membrane vesicles of Escherichia coli {Hoppe et al . (1984) Biochemistry 23, 5610-5616} . In the absence of subunit a, considerably more and different amino acid residues of subunit c are modified . The labelling data are discussed in relation to structural aspects of F0 and functional properties of proteoliposomes reconstituted with F0 or individual subunits.

FEBS Lett, 1988 Jan 4, 226(2), 232 - 4
Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H; Metelev VG et al.; Chimeric oligo(ribo-deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H . It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11.

Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 372 - 6
Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum; Hildebrand M et al.; The rps12 gene in tobacco chloroplasts consists of three exons that code for a polypeptide with homology to Escherichia coli ribosomal protein S12 . C-terminal exons 2 and 3 of rps12 are located in the inverted repeat regions of the tobacco chloroplast genome . Exon 1 of rps12 is 29 kilobase pairs downstream of the nearest copy of exons 2 and 3 and 69 kilobase pairs away from the distal copy of exons 2 and 3 . RNA gel blot hybridization analysis and primer extension sequencing of cDNA to rps12 encoding RNAs indicate that exon 1 and exons 2 and 3 are encoded on separate transcripts . Exon 1 and exons 2 and 3 are covalently ligated in the correct reading frame in rps12 mRNA . These results indicate that a bimolecular (trans-) splicing event occurs during the formation of mature rps12 mRNA.

Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 319 - 23
Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene; Thiede MA et al.; Alkaline phosphatase {ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1} is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues . To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library . The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence . ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP . The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane . The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP . The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene . The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases . Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.

J Clin Lab Immunol, 1988 Jan, 25(1), 41 - 5
5-lipoxygenase inhibitor (AA-861) attenuates neutrophil-mediated oxidative stress on the venular endothelium in endotoxemia; Suematsu M et al.; Neutrophil-mediated oxidative stress on the rat mesenteric microcirculation was studied in the experimental model of endotoxin-induced disseminated intravascular coagulation (DIC) by using an intravital fluorescent microscope equipped with a Silicon Intensifier Target Image Tube camera and luminol-dependent chemiluminescence (ChL) analysis . Leukocytes adhering to the venules were visualized by the injection of acridine orange, a fluorochrome tracer which shows high affinity to white cells . Endotoxin (E . coli, O-111 B4) was administered intravenously at a dose of 2 mg/kg/hour . After starting the infusion of endotoxin, the number of adherent cells gradually increased in the venular endothelium and was followed by a transient neutropenia . ChL activities from neutrophils were also significantly elevated, which may reflect the enhanced ability to generate oxygen-radicals . To elucidate the role of 5-lipoxygenase products in the locomotive and metabolic changes of neutrophils, the effects of AA-861, a specific inhibitor of 5-lipoxygenase was tested . In addition prednisolone and indomethacin were evaluated . AA-861 and prednisolone reduced neutropenia, leukocyte adhesion to the venular walls and ChL activities from neutrophils . It was concluded that 5-lipoxygenase may modulate neutrophil-mediated oxidative stress on microvasculature in endotoxin-induced DIC.

Bioorg Khim, 1988 Jan, 14(1), 43 - 7
{An improved chemico-enzymatic method of synthesizing long DNA segments}; Kobets ML et al.; A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described . A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates . This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment) . It was cloned into an E . coli plasmid vector pBR322 and its sequence confirmed.

Proteins, 1988, 3(1), 18 - 31
Flexibility of the DNA-binding domains of trp repressor; Lawson CL et al.; An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals . Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains . Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand . We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator . This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 131 - 8
{A method of determining the primary structure of oligodeoxyribonucleotides}; Sviriaeva TV et al.; Sanger method was modified to fulfill the requirements of sequencing of oligodeoxyribonucleotides . E . coli DNA polymerase I Klenow fragment was used for all the reactions . The method consists of three steps made in succession in one tube: 1 . Optional hydrolysis of a 5'-labeled oligodeoxyribonucleotide primer in order to get a set of primers of different lengths . 2 . Elongation of the produced set of primers in the presence of a template, natural dNTPs and chain terminating dNTP analogs . 3 . Hydrolysis of the products of the previous step in order to remove the unterminated molecules . Change of steps in achieved just by varying the reaction conditions without any product purification . The method in insensitive to the presence of admixture of oligonucleotides which is not complementary to the primer or to the template.

Folia Microbiol (Praha), 1988, 33(2), 101 - 7
Effect of detergents on aspartate ammonia-lyase activity in Escherichia alcalescens; Malanik V et al.; The effect of twelve detergents on aspartate ammonia-lyase activity of Escherichia alcalescens used for the production of L-aspartic acid was tested . Best permeabilization was found with Triton X-100, Slovafol 910 and Corona, a mixed commercial preparation . In contrast to Triton X-100 and Slovafol 910, a much narrower range of suitable concentrations was observed with Corona.

Curr Genet, 1988, 13(1), 75 - 82
Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants; Falconet D et al.; The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera . All three genes exhibit a high degree of homology except within two variable regions . When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3' end of the gene . The 5' and 3' ends of the wheat mitochondrial gene were determined by S1 nuclease mapping . Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5' region of the 26S rRNA gene or in the 18S rRNA gene.

Am J Vet Res, 1988 Jan, 49(1), 125 - 9
Synergistic effects of Fusobacterium necrophorum lipopolysaccharide, cytoplasmic, and culture supernatant fractions on induction of acute hepatic necrosis in rabbits; Nakajima Y et al.; Synergistic effects of toxic fractions of Fusobacterium necrophorum were examined for evaluation of the role of the toxin in inducing liver abscesses in rabbits . Cytoplasmic and culture supernatant fractions of F necrophorum had preparative activity for the Shwartzman reaction, and lipopolysaccharide of F necrophorum had preparative and provocative activities for the reaction . All 3 fractions were hepatotoxic . Inoculation into the bile duct with each fraction followed by intravenous inoculation with F necrophorum or Escherichia coli lipopolysaccharide had a greater synergistic effect in inducing severe hepatic necrosis than did inoculations with double doses of the cytoplasmic or supernatant fractions of F necrophorum . This synergism may have been attributable to the Shwartzman reaction.

DNA, 1988 Jan-Feb, 7(1), 63 - 70
DNA amplification in vitro using T4 DNA polymerase; Keohavong P et al.; We have evaluated in vitro DNA amplification by polymerase chain reaction using either T4 DNA polymerase or Klenow fragment of Escherichia coli DNA polymerase I . Both polymerases under optimal salt conditions permit efficient amplification of exon 3 of the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene from human genomic DNA and from plasmid containing the HPRT cDNA . DNA sequences amplified from human genomic DNA, using two 20-nucleotide primers flanking the ends of the exon, showed a marked difference between the two polymerases . T4 DNA polymerase yielded only the expected amplified DNA fragment, whereas Klenow fragment produced many lower-molecular-weight bands in addition to the expected DNA fragment . On the basis of the reported fidelity of in vitro DNA synthesis using Klenow fragment and T4 DNA polymerase, it is expected that the latter will create substantially fewer errors during the amplification process . For these reasons, T4 DNA polymerase should be particularly valuable for amplification of sequences present at a very low frequency requiring many cycles of amplification to be detected.

Am J Physiol, 1988 Jan, 254(1 Pt 2), H181 - 6
Improved techniques for cardiovascular monitoring in rats as applied during endotoxemia; Biber B et al.; We describe a new combination of techniques for measurements of systemic blood pressure, central venous pressure, pulmonary arterial (PA) pressure, PA wedge pressure, and cardiac output in the rat . Application of the method to the conscious rat in a septic shock (Escherichia coli endotoxin iv injection) model demonstrated a response pattern of decreased cardiac output and stroke volume, increased total peripheral vascular resistance and heart rate, and transiently decreased systemic arterial pressure . In the pulmonary circulation, a very brief hypertension and a sustained increase in pulmonary vascular resistance were observed, but changes in PA wedge pressure were small . The soft PA catheter (0.3 mm ID, 0.6 mm OD) had no undue effects on cardiovascular function . We suggest that this combined technique could be useful for many cardiovascular studies in the rat, not only as related to shock research.

Am J Physiol, 1988 Jan, 254(1 Pt 1), E45 - 51
Regional lactate production in early canine endotoxin shock; van Lambalgen AA et al.; High serum lactate may not reflect the severity of endotoxin shock: the lactate load could even be formed immediately after the endotoxin challenge . During the first 30 min after endotoxin injection (Escherichia coli; 1.5 mg/kg iv) into anesthetized dogs (4 mg.kg-1.h-1 etomidate, n = 19) we studied arterial lactate concentration; contributions of portal and splanchnic (n = 6), renal and pulmonary (n = 7), and femoral (n = 6) vascular beds to the early lactate rise; and regional O2 extraction and blood flow (microspheres) . In control dogs (n = 5, no endotoxin), we found no significant hemodynamic and biochemical changes . Endotoxin caused an immediate decrease in blood pressure, cardiac output, and organ perfusion, followed by recovery after approximately 5 min to approximately 75% of preshock values at t = 30 min (except for renal blood flow, which remained low) . Arterial lactate concentration started to increase almost immediately after endotoxin and increased rapidly until t = 15 min (to 300%) and then leveled off, but in spite of the hemodynamic recovery it remained elevated . A major part of the early increase in lactate concentration can be explained by splanchnic lactate production . The total splanchnic bed released more lactate than the portal bed, indicating that the liver produces lactate . We conclude that the lactate concentration later in canine endotoxin shock depends on events that occur during early shock in which the liver may play a crucial role.

J Surg Res, 1988 Jan, 44(1), 73 - 81
Naloxone requires circulating catecholamines to attenuate the cardiovascular suppression of endotoxic shock; Allgood SC et al.; The opiate antagonist naloxone (NAL) improves cardiovascular performance in canine hemorrhagic and endotoxic shock . If the release of neural and adrenal catecholamines is attenuated, NAL does not produce the expected improvement in cardiovascular function in canine hemorrhagic shock . This study tests the hypothesis that an endorphin-catecholamine interaction at the heart is responsible for a part of the cardiovascular depression of endotoxic shock . Two groups of five dogs were instrumented to measure mean arterial pressure (MAP), the first derivative of left ventricular pressure over time (LV dP/dt max), cardiac output, and heart rate (HR); they were then subjected to bilateral adrenalectomy and given chlorisondamine to produce ganglionic blockade . At t = 0 min the dogs were given Escherichia coli endotoxin at 1 mg/kg (LD80) . Group I animals received NAL at 2 mg/kg + 2 mg/kg.hr iv from t = 30 to t = 60 . At t = 45 these animals were treated with epinephrine (EPI) at 20 micrograms/kg.hr iv until t = 60 . Group II animals got EPI from t = 30 to t = 60 and NAL from t = 45 to t = 60 at the same doses as Group I . In Group I, NAL alone had no effect on MAP, LV dP/dt max, or HR . EPI significantly increased (P less than 0.002) cardiovascular parameters with MAP increasing from 52 +/- 7 to 159 +/- 14 mm Hg . In Group II, EPI produced a significant increase in all parameters, and the addition of NAL produced a further significant increase; MAP increased from 37 +/- 3 to 126 +/- 16 mm Hg with EPI and then to 175 +/- 11 mm Hg with NAL . These data support the above hypothesis and indicate that circulating catecholamines need to be present to allow naloxone to reverse the cardiovascular depression in endotoxic shock.

J Bacteriol, 1988 Jan, 170(1), 203 - 12
Gene organization and structure of the Streptomyces lividans gal operon; Adams CW et al.; We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes . Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK . Comparison of the inferred protein sequences for the S . lividans gal gene products to the corresponding E . coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes . Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.

Infect Immun, 1988 Jan, 56(1), 278 - 82
Evidence for the phagocytic transport of intestinal particles in dogs and rats; Wells CL et al.; Fluorescent latex beads of two different colors were implanted into separate intestinal segments in individual dogs and rats . Mesenteric lymph node phagocytes subsequently contained multiple beads of one or the other color but rarely both colors, indicating that intestinal phagocytes transported the latex beads to the draining lymph node . Fluorescent labeled Escherichia coli was implanted into rat ligated intestinal segments, and rare mesenteric lymph node phagocytes subsequently contained fluorescent bacteria, suggesting that intestinal bacteria might be transported in the same manner as inert latex beads.

Circ Res, 1988 Jan, 62(1), 25 - 30
Neuropeptide Y prevents the blood pressure fall induced by endotoxin in conscious rats with adrenal medullectomy; Evequoz D et al.; Neuropeptide Y (NPY) is a vasoconstrictor peptide known to be present in the adrenal medulla, the terminal nerve endings, and in plasma . This study was designed to test whether NPY could prevent the acute blood pressure fall induced by endotoxin administration . Normotensive rats were subjected to adrenal demedullation on the right side and were either adrenalectomized or sham-operated on the left side . Eight to ten days later, NPY (0.07 microgram/min i.v.) or its vehicle were infused for 95 minutes into these conscious, semirestrained rats . The same experiments were performed with rats that received an infusion of epinephrine (0.1 microgram/min) . These doses of NPY and epinephrine when given alone had no blood pressure effect . During the last 75 minutes of the 95-minute infusion, endotoxin (lipopolysaccharide Escherichia coli 0.111:B4, 10 micrograms/min i.v.) or its vehicle were administered . In rats with an intact adrenal gland, endotoxin failed to induce hypotension . In rats lacking a functioning adrenal medulla, however, endotoxin induced a pronounced mean blood pressure fall of 55 +/- 11.6 mm Hg (mean +/- SEM) . This blood pressure drop could be prevented equally well with NPY and with epinephrine infusion and averaged 11 +/- 2.3 and 16 +/- 2.4 mm Hg, respectively, at the end of the experiment . Additional rats were biadrenalectomized and supplemented with an excess of glucocorticoids and mineralocorticoids . In these rats also, NPY markedly attenuated the blood pressure fall resulting from endotoxemia . These data taken together indicate that in conscious rats with no adrenal medulla, the acute blood pressure fall induced by endotoxin administration is greatly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Tissue React, 1988, 10(1), 7 - 16
Studies on inflammation in mice: dynamics of inflammatory response induced by extract from Escherichia coli and influence of antiinflammatory drugs; Ishibashi Y et al.; The intraperitoneal injection of E . coli extract in the mouse resulted in a biphasic accumulation of leukocytes . The first phase of leukocyte accumulation was based on neutrophil influx in the peritoneal cavity, which occurred 1-2 days after E . coli extract administration . The second phase was based on macrophage influx, which occurred 12 days after E . coli extract administration . Preceding the influx of neutrophils in the first phase, the exudation of plasma proteins in the peritoneal fluid occurred at 4 h . A slight increase in plasma exudation accompanied the macrophage influx in the second phase of leukocyte accumulation . These kinetics of the inflammatory response to E . coli extract were similar to those of the inflammatory response to lipopolysaccharide (LPS), suggesting that the inflammatory response induced by E . coli extract administration was mainly due to the effect of LPS, a component of the E . coli extract . Indomethacin reduced the plasma exudation at 4 h but had no influence on the leukocyte accumulation . On the other hand, dexamethasone suppressed the leukocyte accumulation in both phases.

CRC Crit Rev Biochem, 1988, 23 Suppl 1, S43 - 86
DNA strand exchanges; Griffith JD et al.; Biochemical and electron microscopic studies of the strand exchange reactions catalyzed by the RecA protein of Escherichia coli and the UvsX protein of T4 phage reveal that these reactions proceed in three distinct steps . The first step, termed joining, involves the assembly of RecA (or UvsX) protein onto a single-stranded DNA (ssDNA) molecule and the subsequent search for homology with a double-stranded DNA (dsDNA) partner and formation of a stable synapsis . In the second step (envelopment/exchange), the exchange of DNA strands occurs fueled by the hydrolysis of ATP . The third step (release of products) entails the resolution of the complexes and dissociation of the protein from the DNAs . The structure of the intermediates in the in vitro reactions catalyzed by the RecA and UvsX proteins is emphasized in this review . The results of pairing different DNA molecules in vitro (such as linear ssDNA pairing with linear or supertwisted dsDNA) are described . Paranemic joints represent a major pathway of joining between two DNA molecules which may involve, in some cases, most of the DNA substrate molecules . Since the nature of paranemic joints has only recently begun to be understood, the nature, role, and possible in vivo function of paranemic joining are considered.

Protein Seq Data Anal, 1988, 1(4), 263 - 7
Periodic distribution of homologous genes or gene segments on the Escherichia coli K12 genome; Kunisawa T et al.; A computer search for homologous relationships among Escherichia coli proteins has been carried out with the use of databases . Homologous genes or gene segments thus identified at the amino acid sequence level have a tendency to lie on the genome separated by distances of multiples of 7 min . This relatively regular pattern of gene distribution on the E . coli genome is interpreted as reflecting the early history of multiple genome doublings . Evolutionary advantages of duplications of the total genome are discussed in relation to the acquisition of new multistep pathways such as the Krebs cycle and to the generation of a variety of proteins in existence today.

Gene, 1988, 63(1), 65 - 74
Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli; Strauch E et al.; Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase . A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23 . The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tu494 . Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb Bg/II fragment . This fragment was shown to include the Ptt-resistance promoter . Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations . Biochemical investigations revealed that the Bg/II fragment codes for a Pt N-acetyltransferase.

Gene, 1988, 63(1), 135 - 9
Promoter vectors with restriction-site banks; Bleicher F et al.; New vectors harboring the promoter for the chloramphenicol acetyl transferase gene (cat promoter) have been constructed . These vectors are all derived from pJRD184 {Heusterspreute et al., Gene 39 (1985) 299-304}, which contains a restriction-site bank . The cat promoter has been inserted at various positions and in reverse orientations so that almost all the restriction sites originally present on JRD184 can be used in cloning experiments . The expression of the aceK gene of Escherichia coli cloned under the control of the cat promoter has been tested . A large increase in the synthesis of the isocitrate dehydrogenase kinase, the aceK gene product, has demonstrated the efficiency of the newly constructed vectors.

Arch Geschwulstforsch, 1988, 58(2), 73 - 8
Genotoxicity assessment of the plasmacytomagenic agent pristane (2.6.10.14-tetramethylpentadecane) and four related alkanes by the SOS chromotest; Janz S et al.; The most extensively studied model of plasmacytomagenesis is the induction of plasmacytomas in BALB/c mice by i.p . injections of mineral oil or, chemically more defined, by several branched alkanes such as pristane (2.6.10.14-tetramethylpentadecane), phytane (2.6.10.14-tetramethylhexadecane), and 7-n-hexyloctadecane . The available evidence suggests that the primary biologic action of these plasmacytomagenic agents is to induce the formation of a chronic granulomatous tissue, the histological matrix of plasmacytoma development . However, certain genotoxic effects caused by the presence of these substances can not be ruled out a priori . Pristane, 2-methyldodecane, and 1.3-di-tert-butyl-5-methyl-cyclohexane as well as perhydroanthracene and hexahydrodibenzsuberane were proofed as potential genotoxic agents by the SOS chromotest, a quantitative bacterial colorimetric assay for genotoxins . The substances tested did not express any sign of genotoxicity, but exerted toxic effects to the E . coli tester strain.

Z Naturforsch {C}, 1988 Jan-Feb, 43(1-2), 91 - 8
Selective binding of amino acid residues to tRNA molecules detected by anticodon-anticodon interactions; Bujalowski W et al.; Anticodon-anticodon pairing of complementary tRNA's has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA . This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons . Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNAPhe.tRNAGlu pairing constant Kp, whereas Kp remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide . The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNAPhe . The binding constants evaluated according to this model (Phe-amide 120 M-1, Tyr-amide 160 M-1 and Trp-amide 580 M-1) are consistent with values obtained independently by fluorescence titrations with tRNAPhe . Selective binding of these amino acid residues to tRNAPhe is deduced from the observed concentration dependence which is not compatible with a corresponding binding process to tRNAGlu . Addition of glutamic acid diamide induces an increase of the tRNAPhe.tRNAGlu pairing constant, which is however equivalent to that observed for tRNAPhe.tRNALys pairing and thus does not demonstrate a selective binding to tRNAGlu . The pairing of tRNAPhe with tRNAGlu is strongly enhanced by addition of Mg2+ or spermine . Evaluation of the Mg2+ data by a site model leads to constants of 360 M-1 for the binding of Mg2+ to monomer tRNA and 3000 M-1 for the binding of Mg2+ to the tRNAPhe.tRNAGlu dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Q, 1988 Jan, 10(1), 48 - 52
Therapeutic efficacy of doxycycline hyclate in experimental Escherichia coli infection in broilers; Goren E et al.; Treatment of experimentally induced colibacillosis in broilers with doxycycline hyclate through the drinking water was just effective at a dose of 200 mg/kg body weight (1000 ppm) . The achieved therapeutic effects were similar to those of tetracycline at the same dose and of flumequine at a dose of 19 mg/kg body weight (100 ppm).

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 201 - 8
{Stability of normal, abnormal and recombinant proteins in Escherichia coli strains deficient for intracellular proteinase La--the product of the lon gene}; Ryzhavskaia AS et al.; Escherichia coli Lon-mutants deficient in intracellular protease La have been isolated . The rate of degradation of normal cellular proteins was 1.5-2-fold lower in Lon-mutants as compared with that of the wild type strain . The rate of degradation of canavanine-containing abnormal proteins, as well as foreign proteins was significantly higher in E . coli than that of normal proteins . Lon-mutants possessed 2-2.5-fold lower rates of degradation of abnormal proteins as compared with Lon+-strains . The rate of degradation of human interferon alpha-2 was 10-fold higher in E . coli than that of abnormal proteins . B . amyloliquefaciens alpha-amylase degraded in E . coli with the rate comparable with that of abnormal proteins, since chloramphenicolacetyltransferase from Tn9 was stable in E . coli . The rate of degradation of interferon alpha-2 was 2-fold lower in Lon-mutants (half-life 23-26 min) than in the initial strain (11-12 min) . Lon-mutants were effectively used as recipient strains for constructing strains-producers of several human alpha- and beta-interferons.

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 187 - 94
{Interaction of tryptophanase with substrate analogs}; Zakomyrdina LN et al.; Tryptophanase from Escherichia coli was studied with respect to its interactions with L-alanine, beta-chloro-L-alanine, L-phenylalanine, L-methionine, L-threonine, beta-phenyl-DL-serine (threo form) and also with a new tryptophan analog oxindolyl-L-alanine . Slow transamination of L-alanine in the active site of the enzyme was observed . Some evidence is presented which indicates that the side transamination reaction occurs during incubation of tryptophanase with an adequate substrate, beta-chloro-L-alanine . Absorption and circular dichroism (CD) spectra of the enzyme-quasisubstrate complexes have been recorded . Addition of beta-phenylserine and threonine to the enzyme induces a decrease of absorbance at 337 nm and an increase of absorbance at 420 nm . The spectral changes are associated with inversion of the CD sign, i.e . with disappearance of positive CD in the 420 nm band and appearance of negative CD in this band . It is inferred that beta-phenylserine and threonine form an external coenzyme-substrate aldimine which undergoes slow conversion to give a keto acid and the free enzyme . Addition of oxindolylalanine to tryptophanase results in the formation of an intense narrow absorption band at 504 nm with a shoulder at about 475 nm . This band belongs to a quinonoid intermediate . A positive CD is seen in the 504 nm band; the dissymmetry factor (delta A/A) in this band is much smaller than that in the absorption bands of the free enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 111 - 6
{The role of in vivo interaction of proteins SSB with DNA polymerase II}; Fradkin GE et al.; In order to elucidate the role of in vivo interaction of single-strand DNA binding protein with DNA polymerase II isogenic strains of Escherichia coli were constructed combining the ssb+, ssb-1 alleles with DNA polymerases I or II mutations; their radiosensitivity and a level of UV-induced DNA degradation were studied . Received findings suggest a functional antagonism of SSB-proteins depending on intracellular conditions (from the balance of DNA and protein synthesis) . The SSB-proteins provide the stability of the genome or, vice versa, perturb the stability of the genome, by degrading of DNA macromolecules.

Microbiol Immunol, 1988, 32(1), 115 - 7
Chemotaxis for methamphetamine in Escherichia coli; Fukunaga S et al.; Escherichia coli showed the chemotactic response for methamphetamine, a biogenic stimulative amine . The response was qualitatively detected by using a paper disk filter . The quantitative assay showed that E . coli responded to methamphetamine at 10(-5) M or more.

J Dairy Sci, 1988 Jan, 71(1), 29 - 40
Molecular cloning and expression of bovine kappa-casein in Escherichia coli; Kang YC et al.; A cDNA library was constructed using poly(A) +RNA from bovine mammary gland . This cDNA library of 6000 clones was screened employing colony hybridization using 32P-labelled oligonucleotide probes and restriction endonuclease mapping . The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method . The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein . The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76 . The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively . Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein.

Gene, 1988, 62(1), 135 - 9
Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme; Tsurushita N et al.; Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution . The resulting DNA was transfected into E . coli JM101 without further treatment . Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum . Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis . Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.

Vopr Virusol, 1988 Jan-Feb, 33(1), 37 - 44
{Immunodiagnosis of AIDS using preparations of the recombinant antigen of the surface protein of the HTLV-III virus}; Kovgan AA et al.; A technological scheme has been developed for purification of recombinant antigen of surface protein (ASP) of the causative virus of AIDS from Escherichia coli cells carrying plasmid pL2 which codes for the synthesis of a hybrid polypeptide consisting of phage lambda N protein (59 amino acid residues) and a fragment of SP (env) of HIV virus (569 a.r.) . Purification of ASP is based on two separation principles: fractionation of polypeptides of bacterial lysates by preparative isoelectrofocusing in a granulated gel layer and the method of preparative polyacrylamide gel electrophoresis in "Multiphor" apparatus (Pharmacia) . The ASP purified by these methods was used for construction of an immunodiagnostic preparation for AIDS, employing for this purpose solid-phase enzyme-immunoassay in two modifications: competitive sandwich method and analysis of the tested sera with direct sorption of ASP on nitrocellulose filters.

Prikl Biokhim Mikrobiol, 1988 Jan-Feb, 24(1), 35 - 41
{Enzymatic synthesis of L-malic acid from fumaric acid using immobilized Escherichia coli cells}; Verevkin AN et al.; Optimal conditions were chosen for cultivation of Escherichia coli 85 cells with a rather high fumarate-hydratase activity on a cheap medium containing no edible raw material . An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on E . coli 85 cells immobilized in carrageenan . The enzymatic synthesis of L-malic acid from potassium fumarate was kinetically studied and optimized . Some thermodynamic parameters of fumaric acid hydration into malic acid were determined . A technique for assaying the reaction mixture was developed that involved high performance liquid chromatography.

Mol Microbiol, 1988 Jan, 2(1), 43 - 52
Fumarate reductase of Escherichia coli: an investigation of function and assembly using in vivo complementation; Condon C et al.; Recombinant plasmids which carried portions of the Escherichia coli frd operon were constructed and their expression examined by in vivo complementation of E . coli MI1443 . This strain lacked a chromosomal frd operon and was unable to grow anaerobically on glycerol and fumarate . Introduction of all four fumarate reductase subunits into E . coli MI1443 was essential for the restoration of growth . The FRD A, FRD B dimer (but neither subunit alone) was active in the benzyl viologen oxidase assay . Both FRD C and FRD D were required for membrane association of fumarate reductase and for the oxidation of reduced quinone analogues . Introduction into E . coli MI1443 of the frdABC and frdD genes on two separate plasmid vectors failed to restore anaerobic growth on glycerol and fumarate . Thus separation of the DNA coding for the FRD C and FRD D proteins affected the ability of fumarate reductase to assemble into a functional complex.

Arch Microbiol, 1988 Jan, 149(3), 232 - 9
Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli; Meury J; The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate . The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1-2 min and restore a higher initiation frequency . High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process . It is inferred that turgor could control DNA replication and cell division in two separate ways . Glycine betaine action is not mediated by K+ ions as the internal level of K+ ions is not modified significantly following glycine betaine accumulation.

Zh Mikrobiol Epidemiol Immunobiol, 1988 Jan, (1), 8 - 11
{Kinetic analysis of the action of cetyltrimethylammonium bromide on the membranes of the capsular variant of Escherichia coli CA189 recorded by ethidium bromide fluorescence}; Puchkov EO et al.; A mathematical model, describing the kinetics of ethidium bromide probe fluorescence in the suspension of E . coli CA 189 capsular antigen after introduction of cetyltrimethylammonium bromide (a detergent), has been developed . Experimental testing of this mathematical model has confirmed that it describes the kinetics of the probe fluorescence augmentation sufficiently accurately . The new model permits the quantitative characterization of the rate of surfactant diffusion through the bacterial capsule.

Nippon Geka Gakkai Zasshi, 1988 Jan, 89(1), 6 - 20
{Experimental study on the pathophysiology of endotoxin shock as analysed by alterations in thromboxane B2 and 6-keto-PGF1 alpha levels}; Baba H; To evaluate the pathophysiological role of thromboxane A2 (TXA2) in endotoxin shock, plasma concentrations of TXA2 and PGI2 following E . coli endotoxin (ET) administration were measured in dogs and rats by radioimmunoassay of their stable metabolites TXB2 and 6-keto-PGF1 alpha, respectively . Also, the effects of TXA2 synthetase inhibitor (OKY046) on eicosanoid levels, haemodynamics and survival were assessed . The following results were obtained: 1) Survival rates of the rats given 50 mg/kg of ET were 31% at 12 hrs and 17% at 24 hrs . Pretreatment with OKY046 markedly improved the survival rates . 2) Plasma concentrations of TXB2 were rapidly elevated in untreated control dogs and rats following ET administration, whereas plasma 6-keto-PGF1 alpha levels were gradually elevated . TXB2/6-keto-PGF1 alpha ratio showed an early elevation at 15 minutes after ET administration . The ratio became lower than base line, thereafter . 3) In contrast to the controls, animals pretreated with OKY046 did not exhibit significant elevations in plasma TXB2 levels . On the other hand, plasma levels of 6-keto-PGF1 alpha were not altered by OKY046 treatment . 4) In the control dogs given ET, the early elevations in pulmonary artery pressure (PAP) and reduction in lung compliance correlated with the early elevation in plasma TXB2/6-keto-PGF1 alpha ratio . 5) In OKY046-treated dogs, the early elevation in TXB2/6-keto-PGF1 alpha ratio was not seen and PAP increase and lung compliance reduction were prevented . The results suggest that TXA2 plays an important pathophysiological role in the development of endotoxin shock.

Folia Microbiol (Praha), 1988, 33(1), 59 - 67
Mutation analysis of the receptor for colicins E1-E7 . A pilot study; Smarda J et al.; Thirty eight mutant clones of the colicin indicator strain Escherichia coli K 12 ROW, selected by their insensitivity to any of the colicins E1-E7, were isolated . Comparison of their sensitivity-resistance patterns to colicins E1-E7 enabled us to draw a rough preliminary map of the receptor for E colicins . In this receptor, the highly specific binding site for colicin E1 partially overlaps with the domain shared by all colicins E2 through E7 . A specific binding site of this domain appears to be common for colicins E3 and E6; a part of the E3 and E6 binding site is also common for colicins E4 and E5 and a small, least specific, part also for colicins E2 and E7 . Using colicin assay experiments, the binding capacity of colicin E receptor mutants could be estimated . A decreased, but not completely lost ability of certain mutants to bind colicins E, correlated to their lowered sensitivity to them, was found . Thus the phenomenon of partial colicin resistance was established, showing that colicin sensitivity--resistance is not a qualitative but a quantitative marker.

EMBO J, 1988 Jan, 7(1), 269 - 75
Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA; Hurstel S et al.; Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA . These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation . This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface . The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers . Each half-operator contains seven interfering phosphates . These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target . On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis . We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein . A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain . We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay.

Mol Gen Mikrobiol Virusol, 1988 Jan, (1), 33 - 6
{Recombinations during DNA cloning}; Gurevich AI et al.; RecA-independent recombinations accompanying the processes of plasmids preparation and cloning into Escherichia coli cells are induced within the short direct and inverted repeats of several types.

Genetika, 1988 Jan, 24(1), 13 - 22
{Genetic mapping of the ilv7434 mutation providing threonine deaminase resistance to isoleucine inhibition in Escherichia coli}; Gavrilova OF et al.; Location of previously isolated ilv7434 mutation was determined by use of transductional shortening of the F'14 episome . The ilv7434 mutation causes resistance of threonine deaminase (coded for by ilvA gene) to feed-back inhibition by isoleucine . Another phenotype characteristics of the ilv7434 mutant is the ability to feed a lawn of isoleucine auxotrophs in the cross-streak test . The F'14 strain AB1206 carrying ilv7434 mutation was used as a donor for making transductionally shortened episomes in recA recipient . These shortened F'14 episomes containing variable segments of the ilv cluster were then tested for their ability to transfer ilv7434 phenotype by complementation with ilv recA recipients . The data of complementation test suggest that ilv7434 is situated between ilvD and ilvC genes . One of 20 tested shortened episomes carrying, as shown by complementation test, incomplete ilvA gene was found to transfer ilv7434 phenotype by recombination with ilvA527 recA+ recipient . These data allow to conclude that ilv7434 mutation is located within the ilvA gene.

Artery, 1988, 15(2), 71 - 89
Time-dependent effects of endotoxin on the ultrastructure of aortic endothelium; Lee MM et al.; The aortic endothelium from control and Escherichia coli (E . coli) endotoxin-treated rats and rabbits was examined by transmission and scanning electron microscopy . Following the intravenous injection of endotoxin, the animals were sacrificed at intervals ranging from 1 min to 4 hr . As early as 1 min after endotoxin, there was a widening of the subendothelial space (SES) and an increase in tortuosity of the internal elastic lamina (IEL) . At 5 min, the tortuosity of the IEL increased to a peak value, and the SES showed an increase in the amount of smooth muscle cells (SMC) . Initial endothelial damage occurred 5 min after endotoxin: SEM showed some spindle-shaped endothelial cells starting to peel from the underlying SES, and TEM showed some endothelial cells protruding or arching into the lumen . The new findings in this study are that endotoxin injection a) has a very rapid (less than 15 min) effect on rat and rabbit aortic endothelium, including localized endothelial injuries in the intima, and b) induces ultrastructural alterations also in the SES, IEL and portions of the tunica media . These effects were largely reversed within 1 hr after endotoxin administration, thus indicating that the endothelium and other components of the arterial wall can recover with great speeds.

Am J Vet Res, 1988 Jan, 49(1), 90 - 5
Hemodynamics, plasma eicosanoid concentrations, and plasma biochemical changes in calves given multiple injections of Escherichia coli endotoxin; Templeton CB et al.; Twelve male neonatal calves (39 to 50 kg) were allotted to 3 groups of 4 calves each . All calves were anesthetized with halothane, and then Escherichia coli endotoxin was given intravenously (3 times) and intraperitoneally (3 times) during a 6-hour period . Group-1 calves were untreated, group-2 calves were pretreated with a low dose of flunixin meglumine (1.1 mg/kg of body weight), and group-3 calves were pretreated with a high dose of flunixin meglumine (4.4 mg/kg) . In calves of group 1, the mean systemic arterial blood pressure (MABP) and cardiac output (CO) decreased, but pulmonary arterial pressure increased after the initial intravenous and intraperitoneal injections of endotoxin . In calves of this group, these changes were accompanied by increased plasma thromboxane B2 (TxB2) concentration . During this period, increased plasma TxB2 concentration or hemodynamic changes were not detected in calves of groups 2 and 3 . Only calves of group 1 had altered hemodynamics early in the experiment; however, after 6 hours, calves of all 3 groups had similarly decreased CO and MABP . In calves of the untreated group, plasma 6-keto-prostaglandin (PG)F1 alpha concentration increased steadily from the beginning of the experiment until 3 hours later . The CO and MABP were low at the time when serum 6-keto-PGF1 alpha concentration was high; however, these 2 measurements also were low in treated calves who did not have correspondingly high plasma 6-keto-PGF1 alpha concentration . Regional blood flow analysis did not reveal correlations between prostanoid concentrations and altered blood flow to selected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1988 Jan, 49(1), 77 - 81
Lidocaine treatment of dogs with Escherichia coli septicemia; Hardie EM et al.; Effects of lidocaine on organ localization of neutrophils and bacteria and on hemodynamic and metabolic variables were determined during septic shock in dogs . Twelve anesthetized dogs were infused with 10(10) Escherichia coli/kg of body weight through a portal vein catheter over a 1-hour period . Six of these 12 dogs were treated with 2 mg of lidocaine HCl/kg (6 mg/kg/h) 15 minutes after the bacterial infusion had begun . Six dogs not given E coli (surgical controls) were given saline solution at the same rate as the bacterial and lidocaine infusions . Over 4 hours, nontreated dogs with septicemia developed systemic hypotension, decreased cardiac output, increased portal pressure, increased serum alanine transaminase activity, increased liver extravascular water, increased liver glycogen depletion, and decreased PaO2, compared with control dogs . Accumulations of polymorphonuclear leukocytes and E coli were found in the liver and lungs of dogs with septicemia . Lidocaine treatment did not alter the hemodynamic measurements and resulted in metabolic acidosis and hypoalbuminemia . Decreased numbers of E coli were recovered from the liver of lidocaine-treated dogs, whereas increased numbers of organisms were recovered from the blood . Neutrophil sequestration was increased in the liver, but not the lungs of lidocaine-treated dogs.

J Appl Bacteriol, 1988 Jan, 64(1), 89 - 98
Evaluation of the Anderson Baird-Parker direct plating method for enumerating Escherichia coli in water; Havelaar AH et al.; The direct plating (DP) method for enumerating Escherichia coli in food was adapted for water analysis by membrane filtration and a standardized protocol was described . The DP method was found to give equal or better recoveries of E . coli than a membrane filtration method using 0.1% sodium lauryl sulphate agar; the repeatability of the DP method was markedly better . The necessity to transfer membranes from the non-selective medium tryptone soy agar (TSA) to the selective medium tryptone bile agar (TBA) after pre-incubation for 4 h was considered disadvantageous for practical purposes . A double-layer method, where the membrane filter is placed on a layer of TSA poured over TBA, with incubation in an incubator that automatically switches from 37 degrees to 44 degrees C after 4 h, was found to be an acceptable alternative . Recovery of E . coli and inhibition of competitive flora were equal or only slightly less than for the standard DP method.

Biol Chem Hoppe Seyler, 1988 Jan, 369(1), 39 - 46
Active and inactive forms of hemolysin (HlyA) from Escherichia coli; Wagner W et al.; The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W . & Hedgpeth, J . (1982) J . Bacteriol . 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle . However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase . External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication . The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished . On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA . Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes . Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein . An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product . This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.

Environ Mol Mutagen, 1988, 11(2), 241 - 55
Adaptive response of Escherichia coli to alkylation damage; Volkert MR; Treatment of cells with low levels of alkylating agents for extended periods of time causes them to become resistant to the lethal and mutagenic effects of subsequent high-level challenge treatments with alkylating agents . This increased resistance has been called the adaptive response to alkylation damage and results from the induction of an alkylation-specific DNA repair response . The adaptive response is most efficiently induced by methylating agents and is most effective against the lethal and mutagenic effects of methylation damage to DNA . Four genes have been identified as components of this response, ada, alkA, alkB and aidB . The functions of two of these genes are known . AlkA protein functions as a glycosylase that repairs N3-meA, N3-meG, O2-meT, and O2-meC residues in DNA, and Ada protein functions as an alkyltransferase that removes alkyl groups from O6-meG, O4-meT residues as well as methylphosphotriesters . After it interacts with methylated DNA, Ada protein functions as a positive regulatory element that controls the expression of the adaptive response by stimulating the expression of the ada-alkB operon, and the alkA and aidB genes.

J Clin Microbiol, 1988 Jan, 26(1), 92 - 5
Alkaline phosphatase-conjugated oligonucleotide probes for enterotoxigenic Escherichia coli in travelers to South America and West Africa; Oprandy JJ et al.; Several studies have demonstrated the usefulness of 32P-labeled recombinant DNA probes for identifying enterotoxigenic Escherichia coli (ETEC) . The use of radioisotopes and X-ray development, however, severely handicaps the utility of DNA probes in most clinical laboratories . In this study, enzyme-labeled oligonucleotide probes for ETEC LT (heat-labile toxin) and ST (heat-stable toxin) genes were compared with the standard Y1 adrenal cell and suckling mouse assays for their ability to identify ETEC in a population of American adults experiencing acute episodes of diarrhea in South America and West Africa . The LT probe hybridized with 12% (64 of 529) of the E . coli colonies tested, whereas 11% (57 of 529) were positive by Y1 adrenal cell assay . DNA from 9% (47 of 529) of the E . coli colonies tested hybridized with the ST probe, whereas only 5% (28 of 529) produced ST as measured by the suckling mouse bioassay . For the patient samples tested, correlation between probe and bioassay for LT was 97%, or three discrepancies in 111 patients tested . Overall concordance of the ST probe and bioassay was 95%, or five discrepancies in 111 patients . Enzyme-labeled oligonucleotide probes represent a major advance in the diagnosis of ETEC-associated diarrheal disease and may be used in laboratories with minimal equipment.

J Clin Microbiol, 1988 Jan, 26(1), 149 - 50
Predominance of the ac variant in K88-positive Escherichia coli isolates from swine; Westerman RB et al.; Monoclonal antibodies to K88ac and K88ab were used in enzyme-linked immunosorbent assays on Escherichia coli cultures known to produce K88 pili . A total of 415 K88-positive E . coli isolates from nine states were all found to be the K88ac variant . The cultures tested were isolated during the years 1976 to 1985.

J Clin Microbiol, 1988 Jan, 26(1), 116 - 20
Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections; Hofbauer JM et al.; To evaluate the performance of a serological test for human immunodeficiency virus type 1 (HIV-1) infections based on the use of a recombinant envelope gene-derived protein as the antigen, we caused expression of a 1.4-kilobase fragment of HIV.DNA that codes for the complete gp41 transmembrane protein in an Escherichia coli expression vector and used Western blots (WB; immunoblots) prepared with recombinant material (pEX-41) to detect antibodies to HIV-1 . This test detected all 339 sera which were positive by a combination of conventional serodiagnostic assays and produced no false-positive results with 311 negative samples . Also no false-positive results were obtained with 20 sera from systemic lupus erythematosus patients which had high titers of cross-reactive autoantibodies . In six cases, the pEX-41 WB proved to be more sensitive than individual assays applied on their own, and in five cases it was even more sensitive than a combination of conventional assays . We tested 221 sera in both our pEX-41 WB and a commercially available recombinant enzyme immunoassay (EIA {Abbott}) . The results were identical in 188 cases . A total of 27 sera containing antibodies to gp41 as demonstrated in the pEX-41 WB, as well as the Abbott recombinant EIA, had no antibodies to the recombinant core antigen as measured in the Abbott EIA . However, 25 of these sera did stain the 24-kilodalton band on a WB with purified virus . Six sera that were positive in all of the conventional confirmatory assays and reacted strongly with the pEX-41 WB did not recognize the surface antigen used in the Abbott recombinant EIA . We conclude that the use of WB prepared with recombinant-derived p41 offers a very sensitive and specific method to detect antibodies to HIV.

Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 401 - 5
Disulfide bonds and thermal stability in T4 lysozyme; Wetzel R et al.; Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms . Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding . In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation . In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule . In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride . In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation . These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature . We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding . These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution . The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes . The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.

Proc Natl Acad Sci U S A, 1988 Jan, 85(1), 21 - 5
Characterization of the TPR-MET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases; Gonzatti-Haces M et al.; The proteins encoded by the human TPR-MET oncogene (p 65tpr-met) and the human MET protooncogene (p140met) have been identified . The p65tpr-met and p140met, as well as a truncated TPR-MET product expressed in Escherichia coli, p50met, are autophosphorylated in vitro on tyrosine residues . Using the immunocomplex kinase assay, p140met activity was detected in various human tumor epithelial cell lines . In vivo, p65tpr-met is phosphorylated on both serine and tyrosine residues, while p140met is phosphorylated on serine and threonine . p140met is labeled by cell-surface iodination procedures, suggesting that it is a receptor-like transmembrane protein-tyrosine kinase.

Int Arch Allergy Appl Immunol, 1988, 85(1), 127 - 9
Cloning and expression of DNA coding for the major house dust mite allergen Der p 1 in Escherichia coli; Thomas WR et al.; A cDNA clone coding for the major house dust mite allergen Der p 1 was isolated from a lambda gt 11 library . Its sequence correlates with known amino acid sequences of Der p 1 and it produces a fusion protein which reacts with rabbit anti-Der p 1 antiserum.

Am J Public Health, 1988 Jan, 78(1), 64 - 5
Hemolytic-uremic syndrome: a population-based study in Washington, DC and Baltimore, Maryland; Kinney JS et al.; A population-based study of hemolytic-uremic syndrome (HUS) revealed that 20 child residents of Washington, DC and Baltimore, Maryland were hospitalized with HUS from January 1979 through September 1983 . The number of cases peaked during the summer and fall; none occurred during the winter . Incidence of hospitalized cases was higher in Whites and girls than in Blacks or boys, and the average annual incidence was 1.08 cases/100,000 children less than 5 year old . This study demonstrates that HUS is not unique to the West Coast, as previously suggested.

J Surg Res, 1988 Jan, 44(1), 54 - 61
Effect of glucan on neutrophil dynamics and immune function in Escherichia coli peritonitis; Williams DL et al.; Previous studies from our laboratory have demonstrated that glucan, a nonspecific immunomodulator, modifies the course of murine Escherichia coli peritonitis . The protective effect of glucan was mediated, in part, by macrophages . In the present study, leukocyte dynamics in the peritoneal cavity and peripheral blood of glucan-treated mice following E . coli challenge was examined . Additional studies examined in vitro bone marrow proliferation, as well as phagocytosis and intracellular killing of E . coli by neutrophils following glucan administration . ICR/HSD mice were injected ip with glucan (150 mg/kg) or dextrose (5% w/v) on Days 5 and 3 prior to ip challenge with 1 X 10(8) E . coli . Glucan increased (P less than 0.05) total peritoneal neutrophil numbers prior to and following septic challenge . Examination of peripheral blood revealed that ip glucan treatment in E . coli peritonitis significantly (P less than 0.001) increased the number of circulating neutrophils . Additionally, neutrophils from glucan-treated mice showed increased phagocytosis of E . coli in vitro . Glucan therapy also increased bone marrow proliferation . We conclude that (1) glucan enhances peritoneal neutrophil levels, (2) peripheral blood neutrophils are increased following glucan and E . coli, (3) ip glucan increases bone marrow proliferation, and (4) neutrophils from glucan-treated mice showed enhanced phagocytosis of E . coli in vitro . Thus, the beneficial effect of glucan is mediated not only by activated macrophages, but also by the neutrophilic leukocyte.

J Med Microbiol, 1988 Jan, 25(1), 33 - 9
Experimental Escherichia coli peritonitis in immunosuppressed mice: the role of specific and non-specific immunity; Vuopio-Varkila J; An experimental Escherichia coli septicaemia-peritonitis model was adapted to immunosuppressed mice . The mice were made neutropenic by a sublethal dose of cyclophosphamide, which resulted in a 100-fold increase in their susceptibility to intraperitoneal injection of E . coli O18:K1 . A lethal infection could be prevented by passive immunisation with anti-K1 capsular or anti-O18 LPS antibodies but not with anti-J5 bacterial antibodies . The anti-K1 and anti-O18 antisera were able to increase the LD50 of the E . coli challenge by factors of 50 and 5, respectively . The role of non-specific, lipopolysaccharide (LPS)-mediated resistance to infection was also investigated in this model, in which only long-living phagocytic cells such as macrophages are believed to be functional . Pretreatment of mice with LPS was shown to prevent growth of the bacterial challenge in the peritoneal cavity and blood and to result in a five-fold increase in the LD50 of the challenge strain . These findings suggest an important role for macrophages as effector cells in defence against E . coli infection.

J Med Chem, 1988 Jan, 31(1), 129 - 37
Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase; Taira K et al.; The interaction of dihydrofolate reductase (DHFR) from Escherichia coli with drugs such as methotrexate (MTX) and 2,4-diamino-6,7-dimethylpteridine (DAM) has been studied by means of site-directed mutagenesis, fluorescence spectroscopy, and steady-state as well as transient kinetics . A strictly conserved residue at the dihydrofolate binding site of DHFR, phenylalanine-31, has been replaced with tyrosine or valine to ascertain the importance for binding of this hydrophobic amino acid, which interacts with both the pteridine ring and the p-aminobenzoyl moiety . The first mutation (Phe-31----Tyr) has a minimal effect on the binding of the classical inhibitor, DAM . On the other hand, the second mutation (Phe-31----Val) has increased the dissociation constant of DAM from the DHFR.NADPH.DAM ternary complex over 150-fold (greater than 3 kcal/mol) . The dissociation constant of DAM from the (Val31-DHFR).DAM binary complex was too large to be measured fluorometrically . More importantly, these mutations have decreased the overall tight binding of MTX, from 100- to 140-fold (corresponding to a loss of binding energy of 2.2-2.4 kcal/mol) for the Tyr-31 and Val-31 mutants, respectively . These results indicate that hydrophobic interactions between MTX and DHFR are at least as important as formation of the MTX.DHFR salt bridge in the tight binding of MTX.

J Infect Dis, 1988 Jan, 157(1), 149 - 55
Reliable detection of individuals seropositive for the human immunodeficiency virus (HIV) by competitive immunoassays using Escherichia coli-expressed HIV structural proteins; Dawson GJ et al.; We molecularly cloned the gag and env genes of the human immunodeficiency virus (HIV) and expressed fragments of these genes in Escherichia coli . Using the recombinant core and envelope proteins, we developed two competitive immunoassays (CIAs) . Samples that recognized either the envelope or core proteins were considered positive for antibodies to HIV . This test system was comparable with western blot in detecting antibodies in patients with AIDS or AIDS-related complex that were repeatably reactive in the HIV screening test . All 360 individuals who were positive by western blot were positive by the CIA . A total of 844 samples repeatably reactive by an ELISA screening test were negative both by western blot and by the CIA; 48 samples positive by ELISA, but negative or indeterminate by western blot, were positive by the CIA . Alternate research procedures verified the positivity of these individuals . These data indicate that the CIA described here may be useful as an adjunct or alternative to the western blot.

J Comput Assist Tomogr, 1988 Jan-Feb, 12(1), 118 - 21
CT detection of sacral osteomyelitis associated with pelvic abscesses; Merine D et al.; In three patients the diagnosis of sacral osteomyelitis was made when CT demonstrated intraosseous (two) and intraforaminal (one) gas . Two of the three patients also had radionuclide bone scans, one of which was unremarkable . In the other case, radionuclide scintigraphy greatly underestimated the extent of the disease process when compared with CT . All three patients had contiguous pelvic abscesses as a cause of the osteomyelitis . Although there was a high clinical suspicion for an intraabdominal process, the diagnosis of superimposed osteomyelitis of the sacrum was unsuspected . The detection of intraosseous gas is a pathognomonic, albeit uncommon, manifestation of osteomyelitis . Although the radionuclide bone scan is the method of choice for detecting osteomyelitis, CT should be used as a complementary study in certain patients.

J Bacteriol, 1988 Jan, 170(1), 56 - 64
Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions; Herrero M et al.; Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway . The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced . E . coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence . Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA) . Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane . The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides.

J Bacteriol, 1988 Jan, 170(1), 485 - 8
Flagellin domain that affects H antigenicity of Escherichia coli K-12; Kuwajima G; Escherichia coli K-12 mutants with altered flagellum antigenicity were isolated by introducing random deletions into the flagellin gene . The deletions were identified in the central region of the gene . It is suggested that this region corresponds to the flagellin domain molecule which affects flagellum antigenicity.

J Bacteriol, 1988 Jan, 170(1), 463 - 7
Genetic and molecular characterization of the guaC-nadC-aroP region of Escherichia coli K-12; Roberts RE et al.; The guaC (GMP reductase), nadC (quinolinate phosphoribosyltransferase), and aroP (aromatic amino acid permease) genes of Escherichia coli K-12 were located in the 2.5-min region of the chromosome (muT-guaC-nadC-aroP-aceE) by a combination of linkage analysis, deletion mapping, restriction analysis, and plasmid subcloning . The guaC locus expressed a product of Mr 37,000 with a clockwise transcriptional polarity, and the GMP reductase activities of guaC+ plasmid-containing strains were amplified 15- to 20-fold.

J Bacteriol, 1988 Jan, 170(1), 456 - 8
Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees C; Bryan SK et al.; Escherichia coli cells with the pcbA1 allele and temperature-sensitive DNA polymerase III mutations survived at a restrictive temperature if DNA polymerase I activity was present . The Klenow fragment of DNA polymerase I was capable of supporting cell survival as well.

J Bacteriol, 1988 Jan, 170(1), 416 - 21
Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation; Baldoma L et al.; L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization . Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase . Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions . In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio . The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions . In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change . Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase . Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene.

J Bacteriol, 1988 Jan, 170(1), 330 - 4
DNA sequence of the D-serine deaminase activator gene dsdC; Palchaudhuri S et al.; We have determined the DNA sequence of dsdC, the gene that encodes the D-serine deaminase activator protein of Escherichia coli K-12 . The sequence contains a single open reading frame that terminates in a UGA codon . One the basis of the size of the protein, 33 kilodaltons, and the amino acid sequence encoded by the open reading frame, we identified a likely translation initiation codon 731 base pairs upstream of the translation initiation codon for the divergently transcribed D-serine deaminase gene . There is a broad range of codon usage, not surprising in view of the weak expression of the gene . The N-terminal two-thirds of the activator is arginine-lysine rich and quite polar; the remainder is more neutral . The segment of the protein that seems most likely to have potential to form the helix-turn-helix structure characteristic of DNA-regulatory proteins is located near the end of the polar region . The protein contains a region with significant homology to lambda attB.

J Bacteriol, 1988 Jan, 170(1), 308 - 15
Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin; Kren B et al.; Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized . The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase . In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase . Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele . The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al . (J.-O . Hoog, H . von Bahr-Lindstrom, H . Jornvall, and A . Holmgren, Gene 43:13-21, 1986).

Infect Immun, 1988 Jan, 56(1), 71 - 8
Genetic and physicochemical characterization of the recombinant DNA-derived 47-kilodalton surface immunogen of Treponema pallidum subsp . pallidum; Chamberlain NR et al.; Previous work has established the importance of the 47-kilodalton (kDa) surface immunogen of Treponema pallidum subsp . pallidum (T . pallidum) in the immunopathogenesis of syphilis; the 47-kDa immunogen gene was cloned and expressed in Escherichia coli (M . V . Norgard, N . R . Chamberlain, M . A . Swancutt, and M . S . Goldberg, Infect . Immun . 54:500-506, 1986) . To facilitate additional structural-functional analysis of this protein for immunopathogenesis studies, the recombinant DNA-derived molecule was examined with respect to its genetic expression and physicochemical properties . Subcloning of partial PstI digests of the original 47-kDa antigen-encoding DNA segment localized the 47-kDa antigen gene to a 1.3-kilobase (kb) T . pallidum DNA fragment . A 20- to 100-fold enhanced expression of the 47-kDa antigen was obtained when a 2.85-kb DNA insert containing the entire 1.3-kb structural gene was subcloned into a T7 RNA polymerase-dependent expression vector system . Under these conditions, several derivatives of the recombinant 47-kDa protein possessing different molecular masses were observed that were identical to those previously detected on Western blots of native T . pallidum antigens with monoclonal antibodies . Sarkosyl extraction of E . coli recombinant cell envelopes localized the 47-kDa protein to both the inner and outer membranes of E . coli . The absolute requirement of detergents (N-lauroylsarcosine, 3-{(3-chloramidopropyl)dimethylammonio}-1-propane sulfonate, N-octyl-beta-D-glucopyranoside, or Nonidet P-40) for solubilization of the antigen from E . coli cell envelopes and the observation that the recombinant protein partitioned into the detergent phase on Triton X-114 solubilization were consistent with the fact that it is a hydrophobic, integral membrane protein . Western blots of the 47-kDa antigen purified by immunoaffinity chromatography supported results of previous reports that the 47-kDa protein is specific to pathogenic treponemes.

Exp Cell Res, 1988 Jan, 174(1), 177 - 87
The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis; Tsurugi K et al.; When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X . laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation . In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h . This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes . The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm . Considering the results with enucleated oocytes, we suggest that the X . laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus . By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.

Carcinogenesis, 1988 Jan, 9(1), 183 - 5
Mutation spectrum of the mutagen 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido{1,2-a:3',2'- d}imidazole in Escherichia coli; Hebert E et al.; The mutation frequency and mutation spectrum resulting from 3-N-acetylamino-4,6-dimethyldipyrido{1,2-a:3',2'-d}imidazole (AGluP3) DNA adducts using a previously developed forward mutation assay were established . AGluP3-induced mutagenesis requires the umuC gene product(s) and exhibits similar amounts of base pair substitution and frameshift mutation . Comparison between these results and those obtained with the isosteric amine 2-N-acetylaminofluorene suggests the involvement of deacetylated adduct in the molecular mechanisms of AGluP3-induced mutagenesis.

Semin Surg Oncol, 1988, 4(3), 207 - 9
A phase I-II study of high-dose recombinant human interleukin-2 in disseminated renal-cell carcinoma; Javadpour N et al.; Natural interleukin-2 is a lymphokine with an immunoregulatory function . The recombinant interleukin-2 (rIL-2) is cloned from a human lymphoblastoid T-cell line for this trial . The gene is expressed in E-coli, allowing production of large quantities of rIL-2 . This study is an open label phase I-II trial designed to evaluate the toxicity and efficacy of a rIL-2 in disseminated renal cell carcinoma . After surgical resection of the primary tumor, 3 X 10(6) units/m2 day rIL-2 is administered by 2-hr IV infusions daily for 5 days every other week for a total of eight cycles . At the conclusion of eight cycles of therapy, the patients were re-evaluated for response . Patients with complete and partial responses were eligible to receive an additional eight cycles every other week . Patients demonstrating progressive disease during this study were withdrawn . The toxicities of the high-dose rIL-2 included transient hypotension, pyrexia, malaise, and skin rashes . The transient hypotension responded to fluid replacement, and the other toxicities also responded to symptomatic treatment . The early results in ten patients indicate two partial and one complete response . We have concluded that administration of high-dose interleukin-2 has acceptable toxicities with encouraging response and deserves a phase III study.

Int J Immunopharmacol, 1988, 10(2), 145 - 51
Antibody producing ability of mouse spleen cells after subacute dietary exposure to T-2 toxin; Tomar RS et al.; The effects of T-2 toxin on the antibody producing ability of CD-1 mice after dietary exposure to 0, 2.5, 5 and 15 ppm of T-2 toxin for 29 days was studied . The antibody response against sheep red blood cells, a T-lymphocyte and macrophage-dependent response was suppressed at 2.5, 5 and 15 ppm of T-2 toxin exposure . In contrast, the antibody responses against dinitrophenyl-aminoethylcarbamylmethyl - Ficoll (DNP - Ficoll), a T-lymphocyte independent macrophage-dependent response, and Escherichia coli 0127 (LPS), a T-lymphocyte and macrophage-independent response, were not affected . These results suggest that the inhibitory effects of T-2 toxin on antibody-producing ability after subacute dietary exposure appear to be a direct affect on T-lymphocyte function, possibly involving the T-helper lymphocytes.

Gut, 1988 Jan, 29(1), 35 - 40
Specificity of antibodies secreted by hybridomas generated from activated B cells in the mesenteric lymph nodes of patients with inflammatory bowel disease; Chao LP et al.; Hybridomas have been prepared from active B cells in lymphoid tissue draining lesions of Crohn's disease (CD) and ulcerative colitis (UC), by fusion of fresh mesenteric lymph node suspensions with the murine JK myeloma . Two hundred and fifty nine immunoglobulin secreting hybridomas have been obtained from nine patients . The antibodies have been screened for binding to food antigens, sections of human gut, and bacteria including two unidentified acid fast isolates from CD lymph nodes . Autoantibodies, and antibodies to food antigens implicated by others in the aetiology of CD were rare, comprising 1.2%, and 2.5% respectively . Most donors yielded none of these . Thus neither food antigens nor autoantigens are major antigenic stimuli in nodes draining inflammatory bowel disease . On the other hand between 19% and 83% of supernatants from different donors bound to one or more bacterial genus . The mycobacteria and the CD isolates were amongst the genera to which most antibodies bound, though binding to E coli was more frequent . Significantly more CD than UC derived supernatants bound to BCG . As mycobacteria are not though to be part of the normal bowel flora, the high percentage of hybridomas secreting antibodies which bind to this genus is surprising.

J Clin Invest, 1988 Jan, 81(1), 237 - 44
Dexamethasone inhibition of interleukin 1 beta production by human monocytes . Posttranscriptional mechanisms; Kern JA et al.; Dexamethasone is known to have an inhibitory effect on IL-1 production . To determine the mechanism(s) of this inhibition, adherent human blood monocytes were stimulated with Escherichia coli lipopolysaccharide (LPS) (10 micrograms/ml) in the presence of dexamethasone . Nuclear transcription run-off assays showed that LPS induced IL-1 beta gene transcription two- to fourfold and that this induction was unaffected by dexamethasone exposure (10(-5) M) . The lack of dexamethasone's transcriptional effects was further supported by the absence of any significant change in IL-1 beta mRNA accumulation between LPS-stimulated monocytes exposed or unexposed to dexamethasone, as determined by Northern blot analysis . Posttranscriptionally, dexamethasone was found to have multiple effects: slight prolongation of IL-1 beta mRNA half-life, moderate inhibition of translation of the IL-1 beta precursor, and profound inhibition of the release of IL-1 beta into the extracellular fluid . The data indicate that IL-1 beta is first translated as the 33,000-D pro-IL-1 beta protein, the predominant intracellular form, and the processed to a 17,500-D IL-1 beta protein before or during extracellular transport . The major inhibitory effects of dexamethasone appear to be directed at the translational and posttranslational steps involved in these events.

Carlsberg Res Commun, 1988, 53(4), 233 - 46
Characterization and in vitro expression of the cytochrome b-559 genes of barley . II . In vitro transcription and translation; Krupinska K; The two cytochrome b-559 apoproteins of 9.4 kD and 4.5 kD molecular weight have been expressed in vitro using DNA templates containing either the two genes psbE and psbF in tandem or the individual genes . Transcription with E . coli RNA-polymerase or SP6 RNA-polymerase has been followed by translation in E . coli derived lysates . Simultaneous as well as independent synthesis of the apoproteins is possible . A 9.4 kD in vitro translation product has been identified as apoprotein I by immunoprecipitation with a monoclonal antibody specific for the C-terminal part of the 9.4 kD apoprotein of cytochrome b-559 . The isolated psbF gene directs the synthesis of a translation product with a molecular weight of 4.5 kD corresponding to apoprotein II . Expression of the psbE gene requires the presence of endogenous regulatory sequences 5' upstream of psbE, while this is not the case for psbF . Additional in vitro translation products of 5.7 and 2.4 kD molecular weights are synthesized and probably translated from two reading frames starting with two different out-of-phase ATG codons in the nucleotide sequence of the psbE gene.

Acta Biochim Pol, 1988, 35(4), 331 - 42
Functional states of neutrophils as suggested by whole blood chemiluminescence; Zgliczynski JM et al.; Luminol-enhanced chemiluminescence (CL) of whole blood was examined in order to distinguish between activation states of phagocytic cells . The CL response of these cells was provoked by a phagocytic stimulus--polystyrene particles . Four functional states of phagocytes were proposed: "resting", "stand by", "activated" and "exhausted" . The distinction was done on the basis of extent of the CL response to the particles, time pattern of the process, inhibition of CL by plasma and appearance of spontaneous light emission . Freshly drawn blood of healthy individuals exhibits the "resting" profile of CL, but that of patients with bacterial infection reveals CL patterns ascribed in this paper to the "stand by", "activated" or "exhausted" states of phagocytes . The "stand by", "activated" and "exhausted" behaviour of phagocytes in extravasated blood may be induced by preincubation of blood, stimulation with saline extract of Escherichia coli or N-formyl-Met-Leu-Phe, and by some manipulations involved in preparation of the purified neutrophils.

Res Exp Med (Berl), 1988, 188(5), 329 - 39
Neuroendocrine and cardiovascular responses to high-dose corticosteroid therapy in canine endotoxin shock; Watson JD et al.; The neuroendocrine and cardiovascular responses to endotoxin administration and the effects of subsequent high-dose corticosteroid therapy have been investigated in dogs . Shock was induced in anaesthetised animals by a large bolus of E . coli endotoxin (5 mg/kg) followed by a continuous infusion (2 mg/kg per hour) . One hour after induction of shock, the circulating volume was expanded using a colloidal gelatin solution . Fifteen minutes later, one group of five animals received a bolus of methylprednisolone sodium succinate 30 mg/kg, while a control group of five animals was given an equivalent volume of isotonic saline . The administration of endotoxin produced reductions in mean arterial pressure, cardiac index and left ventricular dp/dtmax, together with increases in systemic and pulmonary vascular resistances . These haemodynamic changes were associated with increases in arterial plasma levels of adrenaline, noradrenaline, cortisol, immunoreactive beta-endorphin and immunoreactive metenkephalin . Cardiovascular improvement followed volume replacement and was associated with reductions in circulating catecholamines . No significant haemodynamic or neuroendocrine changes were demonstrated in the 2 h following steroid therapy.

Res Exp Med (Berl), 1988, 188(5), 319 - 28
Adrenal vein and arterial levels of catecholamines and immunoreactive metenkephalin in canine endotoxin shock and their response to naloxone; Watson JD et al.; The alterations in plasma levels of immunoreactive metenkephalin (ir-metenkephalin) and catecholamines in adrenal vein and arterial blood in response to endotoxin, as well as the effects of subsequent naloxone administration, have been investigated in a canine model . Animals were anaesthetised with alpha chloralose and allowed to breathe spontaneously . The left lumbar adrenal vein was cannulated and an intermittent choke allowed retrograde sampling of the adrenal effluent . Severe shock was produced by the administration of a large bolus of E . coli endotoxin (5 mg/kg) followed by a continuous infusion (2 mg/kg per hour) . One hour after induction of shock the circulating volume was expanded using a colloidal gelatin solution . Thirty minutes later one group of five animals received a bolus of naloxone (2 mg/kg) followed by a continuous infusion of (1.5 mg/kg per hour), while a control group of five animals was given an equivalent volume of isotonic saline . The production of endotoxin shock was associated with marked increases in adrenal vein and systemic levels of adrenaline and noradrenaline . Naloxone administration transiently limited the fall in adrenal vein levels of adrenaline and noradrenaline (P less than 0.05) following volume replacement and was associated with a sustained increase in systemic adrenaline levels (P less than 0.05) . Changes in mean arterial pressure confirmed a significant haemodynamic response to naloxone (P less than 0.05) . Alterations in ir-metenkephalin levels in the adrenal vein closely followed the changes in catecholamines, whereas arterial levels rose progressively and were unaffected by naloxone . We conclude that in canine endotoxin shock the opiate antagonist naloxone can transiently increase catecholamine levels in the adrenal effluent and produce a more sustained rise in systemic adrenaline levels . Moreover, the adrenal medulla is not the only source of circulating ir-metenkephalin.

Nucleic Acids Symp Ser, 1988, (19), 139 - 42
The RNA N-glycosidase activity of ricin A-chain; Endo Y et al.; The RNA N-glycosidase activity of ricin A-chain has been characterized . When rat liver ribosomes were used as substrates, the A-chain cleaved the N-glycosidic bond at A-4324 in 28S rRNA . An apparent Michaelis constant (Km) for the reaction was determined to be 2.6 microM and the turnover number (Kcat) was 1777 min-1 . When naked rRNA was the substrate, the A-chain cleaved the same bond in 28S rRNA but at a greatly reduced rate . The Km value was 5.8 microM . The results suggest that the A-chain has a similar affinity for 28S rRNA in both ribosomes and the naked states . When the deproteinized Escherichia coli rRNA was the substrates, ricin A-chain cleaved a N-glycosidic bond at A-2600 in 23S rRNA which corresponds to the ricin-site in 28S rRNA of rat liver ribosomes, while the A-chain has little activity on 23S rRNA in the ribosomes . The results suggest that ricin A-chain acts directly on RNA by recognizing a certain structure in the molecules . Using the secondary structure models for each species of rRNA, we have deduced a loop and stem structure having GAGA in the loop to be a minimum requirement for the substrate of ricin A-chain.

Dev Biol Stand, 1988, 69, 129 - 38
Parameters for the evaluation of long-term stability of tumour necrosis factor preparations; Geigert J et al.; Escherichia coli-derived Tumour Necrosis Factor (TNF) was formulated in the absence of a protein-carrier, both as a solution and as a lyophilized preparation . By means of three stability-indicating test methods (bioassay, SDS-PAGE, IEF), the long-term stability of these TNF preparations was evaluated . Both preparations showed no change upon storage for 9 months at -70 degrees C and -20 degrees C . Depending upon the test method, different rates of change were detected at elevated temperature . The analysis presented will assist in the design of future TNF reference standards.

Hemoglobin, 1988, 12(5-6), 691 - 7
Restriction primer extension method of labeling oligonucleotide probes and its application to the detection of Hb E genes; Gao QS et al.; A new method for labeling oligonucleotides was developed to obtain high specific activity of radioactive probes . In an oligonucleotide molecule, two sequences were designed . One sequence, the 5', contains 19 nucleotides and serves as a template for probe synthesis . The second sequence, 3', contains a consensus sequence which forms a Pst I site after forming a complementary strand with the primer . In the presence of E . coli DNA polymerase I (Klenow fragment), alpha-32P dNTP and other dNTPs, a radioactive labeled oligonucleotide was synthesized by the primer extension method . After Pst I digestion, the probe was different from its template in length by 4 bp and could be separated from each other on urea-polyacrylamide gel electrophoresis (PAGE) . A radioactive oligonucleotide probe with extremely high specific activity up to 10(10) dpm/micrograms could be obtained by the use of this method . The oligonucleotide probes have been used for the detection of the Hb E mutation in this report.

Dev Comp Immunol, 1988 Fall, 12(4), 749 - 60
Acquired and innate resistance to the haemoflagellate cryptobia salmositica in sockeye salmon (Oncorhynchus nerka); Bower SM et al.; Juvenile sockeye salmon (Oncorhynchus nerka, Fulton River stock) were protected from otherwise lethal challenges with the haemoflagellate Cryptobia salmositica by acclimation to elevated water temperatures (20 degrees C) . Fish treated in this manner displayed increased immunity to C . salmositica and yielded plasma showing enhanced lytic activity against the parasite . The acquired lytic activity was antibody- and complement-mediated . In contrast, a stock of naive O . nerka from Weaver Creek, previously identified as having a high innate resistance to the lethal effects of C . salmositica, also had plasma factors that destroyed the parasite in vitro . This anti-Cryptobia activity also involved complement because 1) it resulted in lysis of the parasite, 2) it was heat-labile (40 degrees C for 20 min), and 3) it was largely removed from the plasma by substances capable of activating (binding) complement by the classical pathway (an antigen:antibody complex of Renibacterium salmoninarum and its specific antibody) and the alternate pathway (Escherichia coli lipopolyssacharide) . The complement-mediated lysis associated with innate resistance was apparently the result of activation by the alternate pathway because it occurred in fish lacking antibodies against the parasite . The reaction was unusual in that a long incubation period (about 2 days) was required for maximum lysis of the parasite . At least one component of the innate lytic system depended on disulphide bonds because lytic activity was destroyed by 2-mercaptoethanol.

J Ocul Pharmacol, 1988 Fall, 4(3), 259 - 67
Effects of a fish oil dietary supplement on endotoxin-induced ocular inflammation; Rubin RM et al.; We compared the effects of fish oil versus corn oil dietary supplements on two rabbit models of uveitis induced by either intravenous (IV) or intravitreal (IVT) Escherichia coli endotoxin . Addition of fish oil to a standard diet consistently diminished the rise in aqueous humor prostaglandin E2 levels 24 hours after IVT endotoxin injection or 3 hours following IV endotoxin injection . Aqueous humor thromboxane B2 levels following IV or IVT endotoxin injection were also lower in rabbits fed a fish oil supplemented diet . However, the fish oil diet resulted in only a modest attenuation of increases in ocular vascular permeability following either IVT or IV endotoxin injection . Fish oil supplementation inconsistently reduced leukocyte infiltration into the anterior chamber following IVT endotoxin . In contrast to the reported ability of fish oil dietary supplements to reduce corneal inflammation, these models of anterior uveal inflammation do not seem to be altered in a clinically significant manner.

Trans R Soc Trop Med Hyg, 1988, 82(6), 885 - 9
Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schistosoma japonicum in mice; Mitchell GF et al.; Mice immunized with purified antigen preparations produced in Escherichia coli and containing the glutathione S-transferase (GST) isoenzyme of Schistosoma japonicum (Sj26) can be partially resistant to infection with this parasite . Maximum resistance was approximately 50% and no protection was obtained in BALB/c mice, known low responders to Sj26 . Although only Freund's complete adjuvant has been used, the data obtained indicate that satisfactory levels of resistance to S . japonicum will not be attained by vaccination with Sj26 alone . Other antigens, including the additional GST isoenzyme of S . japonicum Sj28, will probably be required to establish whether Sj26 will be an important component of a defined multivalent vaccine against schistosomiasis japonica.

Free Radic Biol Med, 1988, 5(5-6), 393 - 402
Molecular genetics of superoxide dismutases; Touati D; Molecular genetics of SOD has been recently developed primarily due to the new biotechnologies . Different types of isoenzymes have now been cloned and sequenced from several species ranging from bacteria to human and plants . Knowledge of the nucleotide sequences permitted refinement of structural models and provided information on subcellular locations . Cloned genes allowed the production of large amounts of SOD . They have been used for physiological and regulation studies, structural and enzymatic analyses, and are vital tools for the isolation of mutants . Isolation of mutants is generally essential to the understanding of the biological function of the gene in question . Indeed, SOD deficient mutants have now been isolated in bacteria and yeast . Their properties support, at numerous levels, a major role of SOD in cellular defense against oxygen toxicity . Few data are presently available on the molecular basis of mechanisms that regulate the expression of SOD.

Int J Rad Appl Instrum B, 1988, 15(5), 511 - 5
Labeled polymorphonuclear leukocytes: a comparison of methodology; Chowdhury S et al.; Polymorphonuclear neutrophilic granulocytes were separated from anti-coagulated whole blood using three techniques . The methods employed included volex sedimentation (VS), volex sedimentation with hypotonic lysis (VSHL), and Ficoll-Hypaque gradient separation (FH) . The cells were labeled with 111In-oxine and 111In-tropolone . Studies were done with both blood from normal human volunteers and with canine blood . From the cell counts and differential, the harvested granulocytes, platelets, and red blood cells per milliliter of whole blood were calculated . Using the granulocyte chemotactic response to E . coli in agarose plates, the ratio of chemotactic migration to random migration (c.m./r.m.) was determined . Survival time for 111In labeled granulocytes were also determined in a canine model . The studies demonstrated that all procedures yielded 100% viability by the Trypan blue exclusion test . Chemotactic migration and leukocyte survival times were similar amongst all techniques . With the VSHL technique, there were significantly fewer red blood cells and platelets in the final preparation approaching the results of FH separation . The results suggest that for a relatively pure granulocyte preparation VSHL is an acceptable alternative to FH.

Adv Exp Med Biol, 1988, 242, 135 - 41
Microcirculatory disturbances in endotoxin-induced disseminated intravascular coagulation . The effects of heparin and gabexate mesilate on locomotive and metabolic changes of neutrophils; Suzuki M et al.; Neutrophil-mediated oxidative stress on the rat mesenteric microcirculation was studied in the experimental model of endotoxin-induced disseminated intravascular coagulation (DIC) by using an intravital fluorescent technique and luminol-dependent chemiluminescence (ChL) analysis . Leukocytes sticking to the venules were visualized by the injection of acridine orange, a fluorochrome tracer which shows high affinity to white cells . Endotoxin (E coli, O-111B4, Difco, USA) was infused intravenously at a dose of 2 mg/kg/hr . After starting the infusion of endotoxin, the number of sticking cells were gradually increased on the venular endothelium followed by a transient neutropenia . In order to investigate the distribution of infused endotoxin in the microvasculature, FITC-labeled endotoxin (Sigma, USA) was used . After administration of FITC-endotoxin, multiple patches of fluorescence along the venular walls were observed, while no fluorescent conjugates were found at the sticking neutrophils and along the arteriolar walls . ChL activities of neutrophils were also dramatically elevated, which may reflect the enhanced ability to generate oxyradical species . To investigate the inhibitory effects of heparin sodium and gabexate mesilate which was a synthetic protease inhibitor on locomotive and metabolic changes of neutrophils induced by endotoxemia, both agents were administered prior to endotoxin infusion . Gabexate mesilate attenuated these changes, but heparin sodium did not show any improving effects . It was concluded that endotoxin primarily affects the venular endothelial cells, resulting in the activation of neutrophils . Gabexate mesilate was more likely to attenuate neutrophil-mediated oxidative stress on microvasculature in endotoxin-induced DIC than heparin sodium.

Prog Clin Biol Res, 1988, 284, 105 - 23
The location, modification, and function of the fushi tarazu protein during Drosophila embryogenesis; Krause HM et al.; The fushi tarazu (ftz) protein of Drosophila is required during embryogenesis for the process of body segmentation . In order to study the biochemical properties of the ftz protein, ftz cDNA was expressed in E . coli, and the protein purified to homogeneity . Polyclonal antibodies raised against the purified protein were used to localize and quantitate the protein during embryogenesis . Three temporally and spatially distinct phases of expression were observed, which include a previously undetected period later in embryogenesis . During this last phase, the protein is localized in the developing hindgut . Analysis of embryonic ftz protein on Western blots permitted us to approximate the number of protein molecules per nucleus . During the blastoderm phase of development, when ftz protein is most abundant, we calculate that there are 15,000 molecules of protein per ftz-expressing nucleus . Since embryonic ftz protein migrates more slowly on SDS polyacrylamide gels than protein expressed either in E . coli, or in vitro in a reticulocyte lysate system, it is apparently modified in the embryo . Two-dimensional gel electrophoresis followed by Western blotting resolves the protein into a series of isoforms which have variable charge and electrophoretic mobility . When compared in its ability to bind DNA in a sequence-specific manner, it was also found that ftz protein partially purified from embryos binds with greater specificity than its bacterially expressed counterpart . In this paper, we demonstrate that embryonic ftz protein binds to a specific region within the ftz enhancer element . The potential relationship between these observations is discussed.

Oncogene Res, 1988, 3(3), 293 - 8
Negative regulation of c-abl tyrosine kinase by its variable N-terminal amino acids; Wang JY; Activation of the oncogenic potential of c-abl proto-oncogene has been correlated with the activation of its tyrosine kinase activity . The oncogenes derived from c-abl, e.g., gag/v-abl in Abelson murine leukemia virus or bcr/abl in chronic myelogenous leukemia, lack N-terminal coding sequences of the normal c-abl gene . In mouse and human cells, two sets of N-terminal amino acids encoded by 5'-variable exons are found in c-abl proteins . To assess the importance of N-terminal deletion in the activation of c-abl tyrosine kinase, a full length or an N-terminal deleted c-abl protein was expressed in bacteria and in monkey COS cells . Measurements of the autokinase activity of these two c-abl proteins showed that deletion of the N-terminal amino acids led to a three to five fold increase of the c-abl tyrosine kinase activity . Thus, the N-terminal deletion is important in the activation of c-abl proto-oncogene.

Biomater Artif Cells Artif Organs, 1988, 16(1-3), 227 - 35
Effects of stroma-free hemoglobin solutions on isolated perfused rabbit hearts and isolated perfused rat kidneys; Vogel WM et al.; "Stroma-free" hemoglobin solutions (SFH) cause hemodynamic alterations indicative of vasoconstriction . We studied vasoconstrictor activity in isolated rabbit hearts and rat kidneys of unmodified SFH and of SFH modified by pyridoxylation or glyoxylation, with or without glutaraldehyde cross-linking . The purity and chemical composition of the solutions, all prepared by other laboratories, were not characterized by us . In isolated hearts SFH prepared by conventional methods had potent vasoconstrictor activity . Pyridoxylation or purification by ion exchange chromatography did not alter the constrictor activity . Decreased constrictor activity was observed with human SFH cross-linked by glutaraldehyde treatment, or purified by affinity chromatography, and with bovine SFH purified by ultrafiltration and preparative HPLC . In isolated kidneys modified and unmodified SFH increased renal vascular resistance and decreased glomerular filtration rate with no morphologic evidence of tubular damage.

Proteins, 1988, 3(4), 256 - 61
Unusual zymogen-processing properties of a mutated form of prochymosin; McCaman MT et al.; Site-specific mutagenesis of the gene encoding bovine prochymosin was used to produce a mutated zymogen in which seven contiguous amino acids of the N-terminal propeptide had been deleted and an eighth residue had been substituted . This altered region spans the normal site of autocatalytic proteolysis that occurs at the same time as (enzymatic) activation of prochymosin at acidic pH . Activation of the mutated zymogen at pH 4.5 was extremely slow, and cleavage occurred at an unusual Ser-Lys bond in the propeptide of the zymogen . The mutated prochymosin incubated at pH 2 generated the usual pseudochymosin by cleavage of the normal Phe-Leu bond, but at a rate severalfold slower than the authentic zymogen . These results indicate that even after deletion of seven of 42 amino acids of the propeptide the mutant protein could still assume a prochymosin (zymogen) structure, although these changes did result in striking differences in acid-catalyzed activation and processing reactions at one but not the other of the two processing sites of prochymosin.

Biochimie, 1988 Jan, 70(1), 109 - 17
Expression of the bovine hypothalamic hormone oxytocin precursor in Escherichia coli; Norenberg U et al.; The bovine oxytocin precursor was expressed in Escherichia coli as a fusion protein by cloning the hormone encoding cDNA in frame behind the replicase gene of the RNA phage MS2 . By step-wise extraction with different urea concentrations, the fusion protein was enriched in the 7 M urea fraction and further purified by Sephacryl S-300 chromatography . The oxytocin precursor was cleaved off the fusion protein by cyanogen bromide treatment, chromatographed on FPLC columns and identified by Western blot analysis, using antibodies raised against neurophysin.

J Recept Res, 1988, 8(1-4), 467 - 80
Cell receptor specific targeted toxins: genetic construction and characterization of an interleukin 2 diphtheria toxin-related fusion protein; Murphy JR et al.; We have genetically replaced the diphtheria toxin receptor binding domain with a DNA insert encoding the T-cell growth factor interleukin-2 (IL-2) . The toxin-related IL-2 fusion gene encodes a 70,586 dalton protein, pro-IL-2-toxin . The mature form of IL-2-toxin is exported to the periplasmic space of recombinant Escherichia coli and has a molecular weight of 68,086 . IL-2-toxin has been partially purified from periplasmic extracts of recombinant E . coli, and it is shown to contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components . The chimeric toxin is targeted toward IL-2 receptor bearing T-cells in vitro.

Gene, 1988, 63(1), 1 - 9
The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein; Mayfield JE et al.; Brucella abortus is the causative agent for brucellosis in cattle and man . Development of a single diagnostic test for the differentiation of vaccinated from infected animals and the development of a nonviable 'subunit' vaccine are top priorities of the brucellosis research program in the United States . Preliminary evidence previously showed that a purified 31-kDa protein (thought to be localized at or near the bacterial cell surface) protects against experimental brucellosis in rodents . The gene for this 31-kDa protein has now been cloned in Escherichia coli . The protein is expressed well, apparently from its native promoter, when placed in several different E . coli plasmids . The nucleotide sequence of the flanking and encoding sequences has been determined, and comparison with the N-terminal amino acid (aa) sequence of the mature protein indicates the presence of a putative 28-aa signal sequence . The availability of the 31-kDa protein free of Brucella contaminants now allows rigorous study of the immunological properties of this protein.

Gene, 1988, 62(1), 127 - 34
Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene; Kolar M et al.; An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phleomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans . Transformation frequencies of up to 20 transformants per microgram of DNA were obtained with the ble system . With the amdS marker the frequency was up to 120 transformants . Cotransformation was very efficient when using amdS as a selection marker . The introduction of pAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A . nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P . chrysogenum . A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the beta-galactosidase fusion protein.

Gene, 1988, 62(1), 101 - 9
Identification of the region of cyanobacterial plasmid pDU1 necessary for replication in Anabaena sp . strain M-131; Schmetterer G et al.; Shuttle vectors based on plasmid pDU1 from Nostoc sp . strain PCC7524 are able to replicate both in Escherichia coli and in strains of Anabaena and Nostoc spp . Derivatives partially deleted in the pDU1 portion were tested for their ability to replicate in Anabaena sp . strain M-131 . Plasmid pRL6HE containing a 1.75-kb HindIII-ScaI fragment of pDU1 replicated stably in that cyanobacterium and also in Anabaena sp . strain PCC7120 . Plasmid pRL6HC, containing an even smaller HpaI-ScaI fragment (1.3 kb) replicated in Anabaena sp . strain M-131 but not in Anabaena sp . strain PCC7120 . Similarly, when the 1.75-kb fragment of pDU1 was transferred from pRL6HE to another vector (pRL40 delta), the resulting plasmids replicated in Anabaena sp . strain M-131 but not in Anabaena sp . strain PCC7120.

Mol Microbiol, 1988 Jan, 2(1), 31 - 41
Divergent effects of a dnaK mutation on abnormal protein degradation in Escherichia coli; Keller JA et al.; Escherichia coli bacteria produce at least one 70 kD stress protein, the product of the dnaK gene . We have compared the rates of degradation of different types of abnormal proteins in null Ion E . coli with a partial deletion of the dnaK gene with the rates observed in null Ion dnaK+ cells . We have found that both canavanyl proteins and puromycyl polypeptides are degraded more slowly in the null dnaK mutants than in the dnaK+ strain . However, a temperature-sensitive mutant LacI protein is degraded more rapidly in the null dnaK strain . The stability of this temperature-sensitive LacI protein was also examined in detail under various other conditions.

Gene, 1988, 62(2), 187 - 96
Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus; Leskiw BK et al.; The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S . clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe . The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917 . When the S . clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S . clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity . The protein coding region of the S . clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.

Res Exp Med (Berl), 1988, 188(1), 59 - 66
Early and late platelet sequestration in different organs during endotoxic shock; Al-Sarraf AA et al.; Using a sheep model, platelet sequestration in lungs, liver, kidney, spleen, and brain was studied after the injection of Indium-111-oxine-labeled platelets and E . coli lipopolysaccharide . Immediately following endotoxin-induced shock, platelet sequestration was observed in lungs, liver, and kidney with corresponding decrease in measured Indium-111-oxine platelets in spleen and circulating blood . There was also a decrease in platelet counts as measured in a phase contrast microscope . The early platelet sequestration in lung, liver, and kidney was followed by platelet disaggregation and another phase of platelet sequestration which continued until the death of the animal . No platelet sequestration was observed in the brain.

Mol Gen Genet, 1988 Jan, 211(1), 102 - 5
The yeast ARGRII regulatory protein has homology with various RNases and DNA binding proteins; Messenguy F et al.; Three regulatory proteins are involved in the post-transcriptional control of arginine metabolism in Saccharomyces cerevisiae: ARGRI, ARGRII and ARGRIII . The 880 amino acid ARGRII protein, like some DNA binding proteins, possesses in its N-terminal sequence a cysteine-rich region that presents homology to the zinc binding region of Escherichia coli aspartate transcarbamylase . ARGRII also has a region of 90 amino acids that is 30% homologous to the E . coli ARGR repressor . Moreover a 87 amino acid long sequence of ARGRII contains three stretches with significant homology to some viral, bacterial and pancreatic RNases . We propose a model in which the RNase-like sequence could regulate the expression of arginine anabolic messenger RNAs.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 Jan, 53(1), 95 - 102
Altruistic cell suicide in relation to radiation hormesis; Kondo S; The high radiosensitivity to killing of undifferentiated primordial cells (Bergonie and Tribondeau 1906) can be described as a manifestation of the suicide of injured cells for the benefit of an organism as a whole if their suicide stimulates proliferation of healthy cells to replace them, resulting in complete elimination of injury . This process is called cell-replacement repair, to distinguish it from DNA repair which is rarely complete . 'Cell suicide', 'programmed death' and 'apoptosis' are terms used for the same type of active cell death . Cell suicide is not always altruistic . Altruistic suicide in Drosophila, mice, humans, plants, and E . coli is reviewed in this paper to illustrate its widely different facets . The hypothesis that in animals, radiation hormesis results from altruistic cell suicide is proposed . This hypothesis can explain the hormetic effect of low doses of radiation on the immune system in mice . In contrast, in plants, radiation hormesis seems to be mainly due to non-altruistic cell death . HORMESIS--'the stimulating effect of small doses of substances which in larger doses are inhibitory' (British Medical Dictionary, Caxton Publ . Co., 1961).

J Neurochem, 1988 Jan, 50(1), 167 - 75
Production of polyclonal antisera to choline acetyltransferase using a fusion protein produced by a cDNA clone; Munoz-Maines VJ et al.; A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli . The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits . Three different antisera were produced that could recognize native Drosophila ChAT with low titer . In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers . The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme . It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons . The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography . Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active . In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C . Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Quad Sclavo Diagn, 1988 Jan-Dec, 24(1-4), 45 - 53
{Isolation frequency of enteropathogenic Escherichia coli in the Patro distric from 1974 to 1986}; Casadei BM et al.; In the Prato district in the years 1974-1986 the isolation frequency of serogropus of enteropathogenic Escherichia coli on 12,251 faeces samples pertinent to infantile diarrhoea has been studied . The research reveals a constant decrease in the isolation frequency of the serogroups considered . An hypothesis is quite difficult but this finding is taking world-wide consistency: furthermore some items are distinctive of the researched district and therefore quite interesting.

Carlsberg Res Commun, 1988, 53(6), 371 - 9
Role of Escherichia coli beta-ketoacyl-ACP synthase I in unsaturated fatty acid synthesis; Siggaard-Andersen M; Two activities were found in E . coli extracts which could complement unsaturated fatty acid synthesis of a cerulenin treated E . coli fatty acid synthetase . One of these is beta-ketoacyl-ACP synthase I, but it is not known whether the other activity represents the previously characterized beta-ketoacyl-ACP synthase II . A mutant strain exhibiting a temperature sensitive unsaturated fatty acid synthetic activity apparently lacked an active beta-ketoacyl-ACP synthase I.

Carlsberg Res Commun, 1988, 53(6), 357 - 70
beta-Ketoacyl-ACP synthase I of Escherichia coli: nucleotide sequence of the fabB gene and identification of the cerulenin binding residue; Kauppinen S et al.; The fabB gene of E . coli encoding beta-ketoacyl-ACP synthase I has been isolated by complementation and sequenced . The enzyme has been purified and its NH2-terminal residues sequenced . Identification of the active site was accomplished by tagging with 3H-cerulenin and radio sequencing of the region . Comparison of the deduced primary structures of the fabB gene product with the FAS2 gene product of Saccharomyces cerevisiae revealed the probable active site in chalcone synthases of higher plants.

Adv Exp Med Biol, 1988, 250, 315 - 30
Regulation of protein synthesis by polyamines; Igarashi K et al.; Present experimental data show that the synthesis of ribosomal protein S1 and PI protein was stimulated greatly by polyamines at the early stage after addition of putrescine in polyamine-requiring mutants of E . coli . No macromolecular synthesis was stimulated at this stage . Polyamine stimulation of the synthesis of these proteins probably plays an important role for cell growth . In polyamine-deficient bovine lymphocytes, protein synthesis became perturbed before RNA and DNA synthesis . Among enzymes concerned with DNA replication, thymidine kinase activity was most strongly influenced by polyamines . The activity in polyamine-deficient cells was only 7% of the level in normal cells . Judging from the amount of thymidine kinase mRNA and its distribution in polysomes, it was concluded that polyamines mainly regulate the synthesis of thymidine kinase at the level of initiation of protein synthesis . A polyamine-free protein synthetic system, established from components of rabbit reticulocytes, consisted of globin mRNA, salt-washed ribosomes, partially purified initiation factors, and pH 5 enzymes . Spermidine added to this system not only lowered the optimal magnesium concentration required for globin synthesis, but it also stimulated the globin synthesis 8- to 10-fold . The optimal spermidine concentration was 0.4 to 0.6 mM, a concentration similar to that in intact rabbit reticulocytes . The ratio of alpha to beta globin chains synthesized in the presence of spermidine and Mg2+ was approximately 1.0, while the ratio in the presence of only Mg2+ was approximately 1.5 . The results strongly suggest that polyamines play an important role in rabbit reticulocyte protein synthesis.

Free Radic Biol Med, 1988, 5(4), 225 - 36
Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli; Davies KJ et al.; E . coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins . Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels . Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312) . Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins . We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron . The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation . Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins . Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts . Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000 . Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E . coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.

Clin Lab Haematol, 1988, 10(4), 397 - 401
Endotoxinaemia in sickle cell disease; Thomson AP et al.; Fifty-nine children with sickle cell anaemia (HbSS) or associated haemoglobinopathies were studied prospectively using a chromogenic Limulus amoebocyte lysate assay to detect circulating endotoxin . The 41 children with HbSS (mean age 8 years 9 months) had more serious disease than the 18 with HbSC disease (n = 14) or HbS-beta-thalassaemia (n = 4) (mean age 7 years 2 months), with a greater degree of splenomegaly, lower haemoglobin, and higher white cell counts, platelet counts and bilirubin values (P less than 0.05 for all) . Twenty-nine children with HbSS had evidence of poor reticuloendothelial function, with red cell pitting of greater than or equal to 2% . Three of these 29 had low levels of endotoxin in plasma (0.12-0.24 endotoxin units (EU)/ml); two were clinically well, one had a painful crisis . Eight of 18 children with other sickle haemoglobinopathies had greater than or equal to 2% pitted red cells; none was endotoxinaemic . Therefore, in 37 patients with reticuloendothelial dysfunction, three were endotoxinaemic; all had sickle cell anaemia . Although not statistically significant, this suggests that endotoxinaemia may occur predominantly in patients with reticuloendothelial dysfunction, and is compatible with the hypothesis that systemic endotoxinaemia can derive from the intestine especially when reticuloendothelial function is depressed.

Biomed Biochim Acta, 1988, 47(9), 915 - 20
{Lipopolysaccharide-specific antibodies in intestinal lavage fluid, bile and serum of mice after oral application of inactivated Escherichia coli}; Schroder R et al.; The formation of antibodies against lipopolysaccharide in the mouse after daily administration of 10(10) killed Escherichia coli M17 (serotype 02:K1) was studied . An IgA response in gut is already established at the first day after an immunization period of 5 days . The kinetics of antibody formation in gut lavage fluid and bile are similar . In comparison lipopolysaccharide-specific IgG antibodies against lipopolysaccharide in serum are raised later.

Acta Microbiol Hung, 1988, 35(4), 383 - 7
Long-term preservation of the enteroinvasive Escherichia coli factor responsible for experimental keratoconjunctivitis; Sourek J et al.; The factor responsible for experimental keratoconjunctivitis (EKC) can be successfully preserved in enteroinvasive Escherichia coli (EIEC) strains for decades . In this respect an adequate freeze-drying technique is essential, whereas the question of protective media appears to be of less importance . The preservation of EKC-producing factor may be associated with the maintenance of some other related properties.

Toxicon, 1988, 26(11), 1047 - 56
Cytoskeletal changes induced in HEp-2 cells by the cytotoxic necrotizing factor of Escherichia coli; Fiorentini C et al.; The effect of the cytotoxic necrotizing factor of Escherichia coli on HEp-2 cells was studied by fluorescence and scanning electron microscopy . This cytotoxin, known for inducing the formation of giant multinucleated cells in several cell lines, caused changes in actin and tubulin organization . The presence of membrane ruffles at the cell border and of numerous thick bundles of actin crossing the cell body, suggests that the factor promotes cell spreading; this probably interferes with cytokinesis, ultimately leading to the formation of very large flattened multinucleated cells . Moreover, the nuclear segmentation observed in treated cells seems to be associated with a rearrangement of actin in the perinuclear region and with the presence of tubulin bundles in proximity to nuclear clefts . Although the primary target is still unknown, these findings suggest that the cytoskeleton is affected accounting for the multinucleation process induced by the factor.

Scanning Microsc Suppl, 1988, 2, 191 - 212
Towards a digital model for an electron-microscope image; Burge RE et al.; An image model is defined based on the boundaries between image regions with different textures and series of descriptions of those textures . Six models of texture are studied under the categories of pixel-based and region-based models . Several techniques for the determination of the unit-cell of textures are presented . The model is applied to the consideration of (a) image correction, (b) the classification of image texture, (c) image enhancement including averaging of detail in periodic specimens, and (d) image data compression . A floating point format, which provides a significant simplification for the Huffman code, is also introduced.

Prog Clin Biol Res, 1988, 282, 11 - 28
Discriminations among unsaturated fatty acids; Lands WE; Several different systems in which unsaturated fatty acids selectively serve an important role provide evidence that the enzymes that select the acyl chains can discriminate among small structural features that were not commonly regarded to be an important feature in explaining how the "good" fatty acids serve their roles . Acyltransferases placing low-melting unsaturated acids in phospholipids do not seem to select the acids for that property . Similarly, the high melting saturated acids seem to be selected for esterification by discrimination of features other than a lack of unsaturation or a high melting point . Finally, the fatty acid essential for optimal growth of young mammals, arachidonate (20:4n-6), is often converted to eicosanoids more rapidly than is compatible with a good quality of life for the adults . In contrast, the n-3 analog, eicosapentaenoate (20:5n-3), does not support such rapid growth and has been classified as non-essential, yet it may moderate the formation and function of n-6 eicosanoids in ways that permit a better chance of survival from eicosanoid-mediated disorders . As a result of attention to presumed functions, we have classified certain fatty acids by using features not actually regulating the enzymatic discriminations by which Nature functions . Such dissonance seems to assure that stories we have been providing about the "appropriateness" of certain membrane lipid structures will be altered by future researchers evaluating the action of lipids in the kinetics of the life and death of a cell.

Methods Enzymol, 1988, 164, 148 - 58
Nuclear magnetic resonance techniques for studying structure and function of ribosomes; Bushuev VN et al.; The following conclusions can be drawn from the use of NMR techniques for studies of ribosomes: 1 . The majority of ribosomal proteins are rigidly fixed within the particles, and the most mobile components in the isolated ribosome are L7/L12 proteins from the large subunit . 2 . Interaction of EF-G with ribosomes results in some changes in ribosomal domains, and, particularly, immobilization of L7/L12 proteins takes place . The changes may pertain to the translocation reaction, since complexes with ribosomes, EF-G, and GTP are functional . The results of these studies using 1H NMR show that structural studies with this technique are limited as only a few proteins express their resonances in the 1H NMR spectra (S1, L7/L12) . At the same time such studies are not exhaustive, since only the simplest samples were studied (ribosomes, the ribosomal complex with EF-G) . Complexes with other ligands (tRNA, EF-Tu) have not yet been studied . It is also possible to enhance the resolution of 1H NMR techniques with the help of deuterated factors, ribosomes, and proteins, and to adapt the use of NMR to other nuclei (e.g., the use of fluorinated labels or incorporation of fluoroamino acids into the proteins) . Many other approaches using NMR in biology have still to be explored . Therefore it is hoped that the use of NMR techniques will prove to be very useful in studies of the different functional steps of protein biosynthesis.

Drug Nutr Interact, 1988, 5(4), 249 - 55
The effect of iron deficiency on the immune response in mice; Blakley BR et al.; Weanling male CD-1 mice were fed low-iron or iron-supplemented diets for 31 days . Mice fed the low-iron diet exhibited typical signs of iron deficiency, which included reduced weight gains (P = 0.0041) and anemia (P less than 0.0001) . The effect of iron deficiency on antibody production, lymphocyte blastogenesis, and sensitivity to endotoxin were evaluated . Antibody production against sheep red blood cells, a T-lymphocyte dependent response, was reduced in iron-deficient mice (P = 0.0067) . In contrast, antibody production against dinitrophenyl-ficoll, a T-lymphocyte-independent response, was not affected by iron deficiency (P = 0.291) . Iron deficiency reduced T-lymphocyte blastogenesis induced by concanavalin A (P = 0.011), but had no effect on B-lymphocyte blastogenesis induced by Escherichia coli lipopolysaccharide (P = 0.662) . These results indicate that the immunosuppressive effects of iron deficiency are related to T-lymphocyte function associated with lymphocyte proliferation and antibody production . A significantly increased susceptibility to endotoxin, a T-lymphocyte-independent response involving nonspecific defense mechanisms, was not observed in iron-deficient mice . Mortality associated with endotoxin was 14.2% in the iron-deficient mice as compared to 35% in the iron-replete mice (P = 0.079).

Ups J Med Sci, 1988, 93(3), 289 - 96
Infected echinococcal cyst . A common cause of pyogenic hepatic abscess; Karavias D et al.; Twenty-one cases of hepatic abscesses treated during a period of four years (from 1981 to 1985) at the University of Patras, Greece, are presented . This material includes 10 cases with abscesses caused by suppurated echinococcal cysts, corresponding to 21% of the total number of 47 cases of echinococcal cysts of the liver treated at our department during the same period . A preoperative diagnosis of the suppurated echinococcal cysts by conventional laboratory methods was not reliable . Because of the high frequency of echinococcal disease in our region and the risk of contamination of the peritoneal cavity from echinococcal parasites if the cyst is punctured, the new therapeutic techniques of treating hepatic abscesses by percutaneous drainage have not been applied . The exclusive method of treatment used was surgical drainage which had a satisfactory outcome and a mortality rate as low as 9%.

Proteins, 1988, 4(3), 173 - 81
Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli; Graddis TJ et al.; Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that form the tryptophan binding pocket . Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid . Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants . The results obtained indicate that replacement of threonine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two- and four-fold, respectively . Replacement of arginine 54 by histidine (RH84) or glycine 85 by arginine (GR85) results in complete loss of tryptophan binding activity . Purified mutant and wild type aporepressors were used in in vitro heterodimer studies . The trp repressor of E . coli functions as a stable dimer . A large number of trp repressor mutants produces defective repressors that are transdominant to the wild type repressor in vivo . The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits . An in vitro assay was developed to detect and measure heterodimer formation . Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels . Aporepressors readily formed heterodimers upon treatment at 65 degrees C for 3 minutes . Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan . Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiol Immunol, 1988, 32(12), 1179 - 87
Expression of antigen genes of Leptospira interrogans serovar canicola in Escherichia coli; Yamaguchi T et al.; Leptospira interrogans serovar canicola DNA was cloned into the plasmid pBR322 and introduced into E . coli . Eight out of approximately 10,000 transformants were found to express antigens of canicola by ELISA including colony ELISA blot test using anti-canicola antiserum . The canicola antigens expressed in the transformants reacted with the antisera against the serovars belonging to Canicola serogroup and other serogroups of L . interrogans . They did not react, however, with the antiserum against L . biflexa (with only one exception) nor with the antiserum against Leptonema illini . Thus, the recombinant DNA technique may provide alternative possibilities for preparing antigens of leptospires.

J Basic Microbiol, 1988, 28(8), 553 - 5
Amplification of different ColE1 plasmids in an Escherichia coli relA strain; Schroeter A et al.; Amino acid starved cells of an E . coli relA strain accumulate a large amount of pBR322 plasmid DNA . In this study ColE1 related plasmids of different copy number and size including a high copy number plasmid mutant of pBR322 were amplified in a relA strain of E . coli K-12 under amino acid limitation in order to determine the upper plasmid level in amino acid starved cells . In all cases we measured a 4 to 6 fold increase of the plasmid copy number in comparison to log-phase cells independent of the size, the number of origins per plasmid molecule or the copy number in log-phase cells . The plasmid copy number in amino acid starved cells varies from about 200 (pBR322-dimer) to about 2000 (high copy number plasmid pERIII-BPL4, see Boros et al . 1986) . Rop+ and rop- plasmids show the same amplification rate under the used conditions.

Drugs, 1988, 36 Suppl 4, 80 - 90
History and rationale of oral rehydration and recent developments in formulating an optimal solution; Farthing MJ; Oral rehydration therapy with glucose-electrolyte solutions has been one of the major therapeutic advances of the century . This alarmingly simple intervention developed from a basic scientific observation in the laboratory, when it was shown that sodium and glucose transport in the small intestine are coupled and thus the presence of glucose in an electrolyte solution promotes absorption of both sodium ions and water . Even more important, sodium/glucose co-transport continues despite the secretory diarrhoea of cholera and enterotoxigenic E . coli and after intestinal damage due to rotavirus . Despite widespread use of the oral rehydration solutions (ORS) recommended by the World Health Organization (WHO), controversy continues about the optimal composition of these solutions . Discussion centres around the sodium and glucose concentrations, the osmolality and whether base (bicarbonate) or base-precursor (citrate) is necessary . Already there is a clear divide between the developing world, where the WHO solution (Na 90, glucose 111 and bicarbonate 30 mmol/L) is widely used, and the industrialised world, where solutions with lower sodium and until recently higher glucose concentrations have been favoured . Recently, attempts have been made to optimise ORS using animal and human model systems before submitting new candidate ORS to clinical trial . Results to date suggest that hypotonic ORS containing 50-60 mmol/L sodium and 90-100 mmol/L glucose produce maximal water absorption . The presence of base or base-precursor appears to offer little with regard to the promotion of sodium and water absorption and its role in combating acidosis remains controversial . Complex substrates such as rice powder and glucose polymers may eventually replace glucose in ORS, since their addition reduces ORS osmolality still further.

Cor Vasa, 1988, 30(6), 400 - 4
Septic complications in patients with permanent pacemakers; Loffler S et al.; Nine patients with implanted pacemakers had the diagnosis of septicaemia to endocarditis . The diagnosis was established on the basis of a repeatedly positive haemoculture . The interval since the first pacemaker implantation to the onset of sepsis to endocarditis was about 5 years . All nine patients had previous reoperation either of the pacemaker or its lead due to decubitus . While, in four patients, the route of infection was a pacemaker lead in its extravascular couse, in 5 patients the source of infection was a lead placed right in the venous system . All patients were treated with ATB according to the antibioticogram . 4 patients had the pacemaker lead extracted . In the remaining five, the pacemaker lead was removed by catheterization . All patients recovered . There is only one way of eliminating infection that caused the sepsis, that is, to remove the foreign body present in the patient - the pacemaker leas in this particular case.

Trans R Soc Trop Med Hyg, 1988, 82(3), 489 - 91
Enteropathogenic and enterotoxigenic Escherichia coli as aetiological factors of infantile diarrhoea in rural and urban Ghana; Agbodaze D et al.; There are 4 recognized classes of Escherichia coli that cause diarrhoeal disease in humans: enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), and enterohaemorrhagic E . coli (EHEC) . Preliminary analysis of enterotoxin production in a rural community in Ghana showed a prevalence of 11.0% LT-ST+, 9.5% LT+ST- and 7.5% EPEC . (LT = heat-labile, and ST = heat-stable, enterotoxin) . The results of a similar study in an urban community in Accra, Ghana, showed 10.9% LT-ST+, 5.9% LT+ST-, 1.6% LT+ST+ and 6.5% EPEC . 14 different serotypes of EPEC were isolated in the urban area, whereas 6 serotypes and two untypable strains were isolated in the rural area . The most common serotype isolated in Accra was 0126:K71 and that from the rural area was 0128:K67 . Serotypes 0143:KXI and 0155:K59 are reported for the first time in Ghana.

Microbiol Immunol, 1988, 32(10), 999 - 1005
Characteristics of a colicin plasmid in Escherichia coli strain B177 isolated from a septicemic calf; Harasawa R et al.; A colicin plasmid in Escherichia coli strain B177 isolated from a septicemic calf was characterized . The colicin type was identified as ColV by using reference ColV producers . The colicin plasmid was labeled with transposon Tn903 and subjected to conjugation . The transconjugants examined suggest that the colicin plasmid confers serum resistance . There was no difference in siderophore utilization ability between the transconjugants and host strain SF800 . Bioassay for siderophore suggests that the colicin plasmid specifies the production of iron-chelating compounds available for the host strain.

Braz J Med Biol Res, 1988, 21(3), 565 - 8
A new mechanism of action of dipyrone: blockade of the release of a nociceptive factor from macrophages; de Campos DI et al.; Rat macrophage monolayers pre-treated with endotoxin release into the incubating fluid a factor (MW greater than 10,000) capable of inducing writhing in mice (MNF) . This release was inhibited by dipyrone (3.5-35 micrograms/ml) but not by indomethacin (0.5-2 micrograms/ml) . Writhing in mice induced by the factor is blocked by dipyrone (0.5-50 mg/kg) and indomethacin (0.5-2 mg/kg) . These results indicate that in addition to the previously described direct blockade of hyperalgesia by dipyrone, this drug may also affect the release of MNF, which induces in vivo nociception through the release of prostaglandin-like substances.

Acta Microbiol Hung, 1988, 35(3), 301 - 5
Alterations in the chemical composition and antigenicity of Escherichia coli O89 lipopolysaccharide and lipid A after 60Co gamma irradiation; Elekes E et al.; The chemical composition and the antigenicity of Escherichia coli lipopolysaccharide (LPS) and lipid A were investigated after irradiation with 150 kGy 60Co-gamma ray . Compared to the original preparations, the irradiated LPS showed a significant reduction in glucosamine, glucose and galactose and a decrease in the phosphate content . The fatty acid components were reduced to a smaller degree . Irradiation induced a reduction in the antigenicity of the polysaccharide part . The amount of glucosamine and phosphate decreased in the irradiated lipid A . The fatty acid content was significantly reduced . The alteration in the chemical composition was not paralleled by changes in the antigenicity of irradiated lipid A.

Nucleic Acids Symp Ser, 1988, (19), 33 - 6
In vitro mutagenesis by incorporation of N4-aminodeoxycytidine 5'-triphosphate; Negishi K et al.; We have investigated the possibility of introducing a new way to carry out in vitro mutagenesis . N4-Aminodeoxycytidine 5'-triphosphate was used in the Klenow enzyme-catalyzed chain elongation of a primer oligonucleotide hybridized to a lacZ alpha region of M13mp2 viral single strand DNA, and the possibility of inducing an efficient, randomly distributed point mutations into this particular genomic region was explored . On transfection of the resulting DNA into E . coli, mutant phages emerged at frequencies up to 1% . Analysis of the DNA sequences of the mutants has shown that single transitions, either A to G or G to A, were induced in a random fashion, thus providing data to show the possibility of using this method for production of mutant proteins having various single amino-acid changes in a defined domain.

Nucleic Acids Symp Ser, 1988, (19), 29 - 32
DNA damage caused by ascorbate in the presence of Cu2+ induces mutations; Kobayashi S et al.; The DNA damage induced by ascorbate in the presence of Cu2+ was analyzed by sequencing, and the mutagenic consequences of damages to plasmid pUC18 lacZ' were assayed in a forward repairing system in E . coli JM109 in vivo . Ascorbate induced two classes of DNA damage in the presence of Cu2+, one being non-base-specific direct strand cleavage, and the other being sequence-specific base modification labile to alkali treatment . Radicals generated from ascorbate hydroperoxide were involved in DNA damaging reactions . Ascorbate and Cu2+ caused mutations in pUC18 lacZ' gene . The mutation frequency by this method was about 10(-4) at 18% survivors when measured as a loss of alpha-complementation . All the mutations found were single-base substitutions that occurred in the structural part of the lacZ' gene . They were predominantly G:C----A:T transitions.

Nucleic Acids Symp Ser, 1988, (19), 165 - 9
The synthesis, cloning and expression of a repeating segment of the circumsporozoite surface protein of Plasmodium falciparum; Armah GE et al.; Repeats of the tetrapeptide (NANP) of the circumsporozoite protein of P . falciparum have been found to be immunogenic and possibly may act as vaccines against malaria infection . The DNA duplex encoding for eight of the (NANP) repeating unit and two of the (NVDP) unit have been synthesized, cloned and expressed in E . coli as a fused protein . The recombinant protein was shown to be immunogenic and to have the antigenic activity of the circumsporozoite.

Dev Biol Stand, 1988, 69, 193 - 7
Consideration of the production methods and safety evaluation of cytokines; Liu DT; Cytokines are natural endogenous substances whose biological effects in humans are little known when given in therapeutic rather than physiologic doses . Yet, there is intense interest in seeking their possible clinical use . While E . coli are effective in making "simple proteins" with few disulfide bonds, mammalian cells are becoming more generally used for the production of "complex proteins" with multiple disulfide bonds and glycoproteins . There appears to be much less concern about the safety of possibly oncogenic residual DNA from transformed cell lines, but viral contamination of products continues to be an active concern . Both physicochemical and biological methods are necessary to establish the identity, purity and potency of biological drugs . For proteins to manifest their proper biological and therapeutic effects in humans, their correct conformation must be maintained throughout production, purification and formulation . Regulating novel biological drugs such as the cytokines might raise new scientific issues that are not currently apparent, but the basic principles involved will be consistent with those used to evaluate other biologics, e.g., sound scientific principles, flexibility, case-by-case approach, good common sense and risk vs benefit assessment.

Dev Biol Stand, 1988, 69, 157 - 68
Construction, purification and biological activities of recombinant human interleukin-2 analogs; Boone T et al.; Fourteen human interleukin-2 (IL-2) analogs have been cloned and expressed in E . coli, starting from a chemically synthesized gene for human IL-2 optimized for expression in E . coli . These analogs were purified to greater than 95% purity as determined by SDS-PAGE, and were measured for biological activity in a 3H-thymidine incorporation assay using an IL-2 dependent murine T-cell line (CTLL) . One analog was made which eliminated the N-terminal 23 amino acids from the protein by replacing one restriction endonuclease fragment with another . This analog, which begins at an internal methionine, had no detectable CTLL activity . Thirteen analogs were constructed using oligonucleotide site-directed mutagenesis . Four of these analogs were truncated at various residues near the C-terminus (residues 106, 116, 121 and 126) . These analogs had at least 500-fold lower CTLL activities than the natural recombinant IL-2 . The remaining nine analogs had substitutions at 1, 2, or all 3 of the three cysteine residues in the protein (residues 58, 105 and 125) . Substituting an alanine, asparagine, aspartic acid, or serine at residue 125 resulted in highly active molecules with CTLL activities similar to that of the natural recombinant IL-2 . The analogs with alanine and serine substitutions at residue 125 actually had slightly higher CTLL activities than the natural recombinant IL-2 . Substituting alanine for cysteine at position 125 and serine for cysteine at either position 58 or 105 yielded analogs with about 150-fold lower CTLL activities than natural recombinant IL-2 . Substituting an alanine for the cysteine at position 125 and serines for cysteines at both positions 58 and 105 resulted in an analog with 30-fold lower CTLL activity than the natural recombinant IL-2 . The ten analogs with less than 1.0% of the CTLL activity of natural recombinant IL-2 were tested for competition with the natural recombinant IL-2 by mixing a 10-to 100- fold excess of the analog with the natural recombinant IL-2 and assaying the mixture in the CTLL assay . None of these analog mixtures resulted in a lower activity than mixing the natural recombinant IL-2 with buffer alone, implying that none of these analogs effectively competes with the natural recombinant IL-2 for binding to IL-2 receptors during incubation with the CTLL cells . If reduced binding does occur, it may be the direct cause of their lower activities.

Eur Surg Res, 1988, 20(5-6), 289 - 97
Local pulmonary activation of proteolytic enzymes after Escherichia-coli-induced lung injury in sheep; Andreasson S et al.; Activation of several cascade systems, e.g . coagulation, fibrinolysis, kallikreinkinin, complement and eicosanoid systems, has been implicated in the etiology of septic-lung microvascular injury . A chronic lung lymph fistula preparation in sheep (n = 9) was used to study coagulation, kallikrein-kinin and eicosanoids during Escherichia coli septicemia . Lung lymph flow and lymph composition indicated an increased lung microvascular permeability approximately 2 h after infusion of bacteria . Stable prothrombin and antithrombin III levels in lymph contradicted local activation of the coagulation cascade in the lung and systemic activation was not evident until 4 h after bacteria infusion . Lymph thromboxane B2 and 6-keto PGF1 alpha peaked early (1 h) . Reduced lymph prekallikrein and kallikrein inhibitors indicated local activation of this system in the lung . Systemic activation of kallikrein could not be demonstrated . Thus, (1) changes in systemic blood may not adequately reflect local events and (2) studies of proteolytic enzymes and other inflammatory mediators in lung may contribute to clarifying the etiology of microvascular injury.

Scand J Infect Dis, 1988, 20(6), 659 - 66
Lactoferrin as an indicator of septicemia and endotoxemia in pigs; Gutteberg TJ et al.; The levels of plasma lactoferrin (LF) in response to endotoxin and Escherichia coli infusions in piglets were studied to obtain exact time relation of plasma LF increase in relation to start of endotoxin and E . coli infusions . A new enzyme-linked immunoassay of swine LF is presented . 13 piglets had a 10-fold rapid increase of plasma LF concentrations after 0.25 mg/kg endotoxin intravenous infusion . The initial rise was 3.4 mg/l/h . 14 piglets, receiving 1 x 10(11) E . coli intravenously, showed a higher increase of plasma LF concentrations, amounting to 6-9 mg/l/h . Thus, plasma LF was an early marker of septicemia and endotoxemia.

Physiol Chem Phys Med NMR, 1988, 20(2), 91 - 108
Isolation and characterization of small phosphorylated peptides controlling transcription "in vitro" from trout testis chromatin DNA; Coderoni S et al.; Low molecular weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction . They show very high specific activity in the control of transcription "in vitro" . In this work the biochemical properties of controlling transcription peptide effectors isolated from trout testis DNA are reported . The purified peptides prevailingly contain glutamic acid, serine, aspartic acid, glycine and alanine . Studies of the peptide structure by N-terminal analysis using the dansyl chloride procedure was unsuccessful, suggesting the presence of a blocked NH2 group . At the same time the active peptides cannot be digested by carboxypeptidases . The gel filtration of the chromatin peptidic fractions on Sephadex G-25, Trisacryl GF05 or Sephadex G-15 shows that the active peptides elute as a single major peak with an elution volume corresponding to a molecular weight of about 1000 . The paper electrophoresis performed at different pH and ionic strength shows that the chromatin peptides are separated in two fractions . One of them is strongly acidic and migrates towards the positive pole until pH 1.9, indicating the presence of phosphoric residues which probably exert an important role in the control of transcription "in vitro" . The chromatin peptides are further purified by Sephadex G-10 and high performance liquid chromatography . The amino acid analysis of the purified peptides are reported.

Protein Seq Data Anal, 1988, 1(5), 355 - 61
Regulation in the synthesis of Escherichia coli RNA polymerase proposed from sequence analysis; Otsuka J et al.; A computer analysis of the operons containing Escherichia coli RNA polymerase subunits was carried out to obtain information about the regulation in the synthesis of these subunits, identifying intercistronic promoter and terminator sequences and searching for ribosome-binding sites and possible secondary structures of the corresponding mRNAs . This investigation showed an extensive secondary structure of beta-mRNA, which provides a molecular basis for the mechanism of the phenomenologically known post-transcriptional regulation in the synthesis of beta subunit . Since a similar secondary structure was also recognized in the mRNA corresponding to the upstream part of alpha subunit gene, it is proposed that the synthesis of alpha subunit is autogenously regulated by RNA polymerase itself, probably at the translational level, in the same way as in the synthesis of beta subunit . This regulation not only guarantees the suppression of overproduction of RNA polymerase subunits but also throws light on the problem of how the syntheses of RNA polymerase and ribosome respond similarly to the shift of nutrients and temperature, but differently to the starvation for amino acids.

Comp Immunol Microbiol Infect Dis, 1988, 11(3-4), 189 - 98
Vaccination of pregnant cows with K99 antigen of enterotoxigenic Escherichia coli and protection by colostrum in newborn calves; Valente C et al.; The immune response to the K99 was tested in 45 pregnant cows, subcutaneously vaccinated, for protecting the newborn calves . Serological tests were performed in the blood sera of all animals and in the milk and colostrum sera; hemogram, inhibition of the adhesion to the brush border and histological tests were performed . The calves from vaccinated cows survived the experimental infection after the suction of colostrum in spite of the fact that the calves from control dams died with diarrhea.

Bull Soc Pathol Exot Filiales, 1988, 81(4), 705 - 11
{Escherichia coli in acute diarrhea in a population of infants}; Khemiri F et al.; The incidence of Escherichia coli EPEC and ETEC had been studied on 555 infants from Tunis area . 193 of them do not present any diarrhea . Frequency of EPEC is 9.14% in infants with diarrhea and 3.10% in the group without diarrhea . Analysis of 87 Escherichia coli strains using genetic probes showed that 4 EPEC strains present effacing attachment factor (EAF) and one EPEC strain produce verotoxin . The frequency of ETEC isolated is 18.05%, the majority of them produced ST enterotoxin . Only colonization factor antigen I (CFAI) was found in ETEC strains.

Brain Dev, 1988, 10(6), 365 - 70
Endotoxin, cerebral blood flow, amino acids and brain damage in young rabbits; Ando M et al.; The effects of bacterial endotoxin (lipopolysaccharide; LPS) on the cerebral blood flow (CBF), amino acid levels and brain histology were studied in young rabbits . The CBF was slightly decreased in the cerebral cortex and markedly decreased in the cerebral white matter at 60 and 120 min after LPS administration . Histological examination revealed only slightly pyknotic neurons around small vessels at 24 hours, and multifocal necrosis in the deep cerebral cortex and white matter at 72 hours . Some amino acids were increased in the plasma and brain regions at 24 hours after LPS administration, most of which were essential amino acids . GABA in the cerebral white matter was decreased at 24 hours . At 72 hours, most non-essential and glucogenic amino acids were decreased . These results suggest that the brain histological changes are related mainly to hypoperfusion and vascular damage in the brain . The amino acid changes may also be related to inappropriate amino acid metabolism associated with brain cell damage.

Protein Seq Data Anal, 1988, 1(6), 487 - 98
The interaction of lac repressor headpiece with its operator: an NMR view; Boelens R et al.; Analysis of nuclear Overhauser enhancements in two-dimensional NMR spectra of the complex of lac repressor headpiece with a 14 base pair lac operator fragment shows that the second helix of the headpiece binds in the major groove of DNA, as has been suggested . The orientation of this helix is approximately 180 degrees different from the proposed models and from that found in the X-ray structure of the 434 repressor-operator complex . The model of the lac headpiece-operator complex provides a good explanation for a large amount of biochemical, genetic and physical data.

Free Radic Res Commun, 1988, 5(2), 107 - 15
Iron enhancement of ascorbate toxicity; Higson FK et al.; Iron has been shown to enhance ascorbate-induced damage to both acetylcholine esterase and E . coli B in a manner analogous to previous studies with ascorbate and copper ions . It is suggested that the mechanism of damage entails interaction of iron with biological macromolecules, followed by its reduction by ascorbate . Subsequently, the iron (II) could participate in generating hydroxyl radicals from hydrogen peroxide via the Fenton reaction, which in turn, could damage biomolecules in a site-specific and multiple hit fashion . The high abundance of iron in biological systems, especially in certain storage disorders, may indicate an important toxicological role of the combination of iron and ascorbate.

Crit Rev Biotechnol, 1988, 8(3), 225 - 36
Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants; Sprang SR et al.; We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin . The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine . In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group . At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished . Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195 . Below pH 6, His57 becomes statistically disordered . Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102 . Steric conflict may cause His57 to rotate away from the catalytic site . These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57 . Three experiments were carried out to alter the substrate specificity of trypsin . Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding . While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin . The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates . The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog . In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate . The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity . Structural investigations of these mutants are in progress.

Crit Rev Biotechnol, 1988, 8(3), 159 - 75
Periplasmic proteases of Escherichia coli; Cook RA; In the course of examining the turnover of enzymes and proteins subject to catabolite inhibition and/or catabolite repression in Escherichia coli, we have observed at least three novel calcium- or manganese-activated proteolytic activities restricted to the periplasmic space . The occurrence and level of these proteolytic activities vary with the stage of cell growth and carbon source . Each of these proteases are neutral metalloendoproteases capable of degrading test substrates such as casein, insulin, globin, and protamine and appear to be unique when compared with the known periplasmic proteases in E . coli . One of these proteases (designated protease VII) has been purified to homogeneity and characterized in regard to subunit structure, sensitivity to protease inhibitors and metal ions, and substrate specificity . Immunological and genetic approaches are being employed to determine if these novel proteases arise from a common gene product . The physiological role of these proteases remains to be established.

Lasers Surg Med, 1988, 8(6), 562 - 6
Transport of biologically active material in laser cutting; Frenz M et al.; The transport of biologically active material during laser cutting with CO2 and Er lasers is demonstrated . This transport mechanism removes particles from the surface of gelatin, agar, and liver samples into the depth of the laser-formed craters . The transport phenomenon is explained by a contraction and condensation of enclosed hot water vapor . We show by cultivating transported bacteria in agar that biological particles can survive the shock of the transport . Determination of the numbers of active cells evidences a more pronounced activity of the cultivated bacteria after impact with an Er laser than with a CO2 laser.

J Basic Microbiol, 1988, 28(6), 385 - 8
DNA fingerprinting of conjugative plasmids incompatible with R6K (IncX); Prager R et al.; 14 conjugative plasmids originated from different bacterial hosts and geographical sources were identified as members of the incompatibility group IncX with R6K as the reference plasmid . In spite of their close genetic relatedness (transfer and incompatibility properties), their molecular sizes ranged between 39 and 77 kb and DNA fingerprinting with endonucleases such as EcoRI, EcoRV, BamHI, BglI, BglII, PstI and SalI revealed a great variety of fragment patterns.

Biotechnology, 1988, 10, 61 - 83
Filamentous phages as cloning vectors; Smith GP; The virtue of Ff vectors goes beyond the fact that they deliver a single strand in a convenient package for sequencing and oligonucleotide-directed mutagenesis . Of all vectors in common use they are the easiest to propagate and process . Their genomes can be easily manipulated, and the knowledge acquired over a quarter century of basic research makes their behavior reasonably predictable . For this reason I have emphasized the general properties of Ff phage in this review and dealt at some length with applications that are still not fully developed, I hope this review will inspire readers to continue the tradition of imaginative exploitation of this unique class of viruses.

Digestion, 1988, 41(1), 55 - 60
Peliosis hepatis associated with liver and retroperitoneal abscesses; Van Schil P et al.; A 33-year-old woman was admitted because of coma and severe shock . CT-scan of the abdomen showed the presence of multiple liver abscesses associated with a retroperitoneal abscess which were drained percutaneously . A liver biopsy showed diffuse peliosis . After resolution of the abscesses the patient's general condition improved and she could be discharged from hospital . Peliosis hepatis is an uncommon disorder characterized by dilated sinusoids with formation of blood lakes . Many pathogenetic mechanisms and causal agents have been proposed . Liver biopsy is necessary to establish diagnosis . By withdrawing the offending drug or treatment of the underlying disorder, regression may be observed.

Arch Microbiol, 1988, 150(4), 363 - 7
Temperature-sensitive murein synthesis in an Escherichia coli pdx mutant and the role of alanine racemase; Grogan DW; The basis for disruption of morphogenesis by depletion of pyridoxine derivatives was studied using a pdxH null mutant of Escherichia coli K-12 . Removal of pyridoxal from growing cultures severely inhibited murein synthesis in vivo, whereas simultaneous supplementation with D-alanine effectively prevented inhibition . Extractable alanine racemase was low following such starvation . Selection of mutants overcoming the glycine- or temperature-sensitivity imposed by pyridoxine limitation yielded a variety of phenotypes . The most effective of these extragenic suppressors conferred an elevated alanine racemase activity which was resistant to the effects of pyridoxal removal.

Arch Microbiol, 1988, 150(4), 348 - 57
Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations; Dinnbier U et al.; The sequence of events following the addition of 0.5 M NaCl to cells of Escherichia coli growing in a minimal mineral medium was investigated . Immediately after upshock the cells took up a large amount of K+ and synthesized approximately half the equivalent amount of glutamate concomitantly . After 30 min the cells started to synthesize trehalose, and after 2 h they had replaced most of their initial osmoprotectants by the carbohydrate . Cell trehalose was rapidly replaced by proline, taken up from the medium when added to the osmoadapting cells . The initial rate of this proline uptake was extremely rapid, and with rates observed of up to 0.6 mmol x min-1 x g-1 of cell protein it was approximately ten times faster than that reported in the literature for non-growing cells . These results indicate that for osmoadaptation of growing cells of E . coli the uptake of proline has priority over the synthesis of trehalose, which in its turn is preferred above K+ and glutamate as osmoprotectants . We observed that two mutants with unknown lesions, but which are known to be impaired in osmoadaptation, were inhibited in replacing K+ and glutamate by trehalose, indicating that this is the basis for their defect in osmoadaptation . Further experiments revealed that neither internal pH nor the membrane potential nor the transmembrane protonmotive force are likely to be involved in osmoadaptation in E . coli . However, during osmoadaptation a high internal potassium concentration appeared to stimulate the derepression of proline-uptake systems (mainly system ProP).

Arch Microbiol, 1988, 150(4), 313 - 9
Yeast PAPS reductase: properties and requirements of the purified enzyme; Schwenn JD et al.; The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3'-phosphoadenylsulphate (PAPS) reductase and thioredoxin . The functionally active protein (MR 80-85 k) is represented by a dimer which reduces 3'-phosphoadenylyl sulphate to adenosine-3',5'-bisphosphate and free sulphite at a stoichiometry of 1:1 . Reduced thioredoxin is required as cosubstrate . Examination of the reaction products showed that free anionic sulphite is formed with no evidence for "bound-sulphite(s)" as intermediate . Vmax of the enriched enzyme was 4-7 nmol sulphite.min-1.mg-1 using the homologous thioredoxin from yeast . The velocity of reaction decreased to 0.4 nmol sulphite.min-1.mg-1 when heterologous thioredoxin (from Escherichia coli) was used instead . The Km of homologous thioredoxin was 0.6.10(-6) M, for the heterologous cosubstrate it increased to 1.4.10(-6) M . The affinity for PAPS remained practically unaffected (Km PAPS: 19.10(-6) M in the homologous, and 21.10(-6) M in the heterologous system) . From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second . Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.

J Hyg Epidemiol Microbiol Immunol, 1988, 32(3), 345 - 52
Effect of detergents on pathogenicity plasmids of escherichias; Krivoshein YuS et al.; The study dealth with effects of cationic detergents miramistin (alkylamidopropyldimethylbenzylammonium chloride), catamine AB (alkyldimethylbenzylammonium chloride) and the anionic compound sodium dodecyl sulphate (SDS) on the elimination from E . coli cells of plasmids determining the Hly, Ent and, indirectly, Col, F, and R markers of pathogenicity as well as their transfer upon conjugation . At subbacteriostatic concentrations, miramistin and catamine AB were found to suppress the transfer of Hly, Ent, F, and R plasmids during conjugation when applied to the donor, recipient or added to the conjugation medium without, however, eliminating plasmids . This is due to the disruption by detergents of F, J pili and other surface cell structures resulting in diminished ability to conjugate . Anionic SDS eliminated F and R plasmids without affecting Hly, Ent and Col.

Vet Res Commun, 1988, 12(4-5), 383 - 99
Pathophysiology of diarrhoea induced by a combined infection with transmissible gastroenteritis virus and enterotoxigenic Escherichia coli in newly-weaned piglets and the effect of flurbiprofen treatment; Cox E et al.; In newly-weaned 3-4 week old piglets (n = 29) diarrhoea (100%) and vomiting (65%) were induced by inoculation with transmissible gastroenteritis virus and enterotoxigenic E . coli strains (0(149):K91:K88ac; LT, STa and STb enterotoxin positive) . This combined infection resulted in pronounced mortality within 7 days . During this period the piglets had decreases in body weight, arterial pressure and leucocyte count and increases in heart rate and in total plasma protein concentration . The plasma pH and lactic acid concentration decreased, whereas the values for pO2, pCO2 and frequency of respiration did not change significantly . No significant changes in the serum concentrations of potassium, chloride or calcium were observed, whereas sodium concentration revealed a transient increase . In shocked and dying piglets an increase in haematocrit was observed, whereas base excess and bicarbonate concentration decreased . Flurbiprofen, a potent non-steroidal anti-inflammatory drug, administered intramuscularly on 3 successive days following the combined infection at a dosage of 1 mg/kg/12 h was without beneficial effect on diarrhoea or mortality.

Gene Anal Tech, 1988 Jan-Feb, 5(1), 9 - 15
Efficient in vitro expression of interferon alpha analogs using SP6 polymerase and rabbit reticulocyte lysate; Tymms MJ et al.; We have investigated the use of in vitro expression as a quick and convenient means of screening large numbers of interferon (IFN) analogs generated using in vitro mutagenesis . The IFN-alpha 1 mRNA generated from DNA template using SP6 RNA polymerase is efficiently translated in rabbit reticulocyte lysate (RRL) . The antiviral specific activity of this RRL-synthesized IFN-alpha l is equivalent to the yeast-synthesized protein . In contrast with the yeast-expression system, where some IFN-alpha analogs are poorly expressed, all analogs tested were well expressed in RRL.

Gene Anal Tech, 1988 Jan-Feb, 5(1), 1 - 4
In vivo plasmid construction by integrative recombination within a 156 bp sequence homology; Park SF et al.; pHG165, a pBR322 copy number derivative of pUC8, has a 38-bp polylinker multiple cloning site located at the 5' end of lac alpha . A further 156 bp, 3' to the multiple cloning site, completes the coding sequence for the production of the beta galactosidase alpha peptide . We describe the use of in vivo plasmid-chromosome cointegrates as a construction method that in this instance has enabled us to cross out the pHG165 multiple cloning site to obtain a wild-type beta-galactosidase sequence for the alpha peptide . Because the DNA sequence available for homologous recombination was only 156 bp in length, the frequency of crosses that removed the multiple cloning site was less than 1 x 10(-9) . These crosses were easily obtained, however, after amplification by ampicillin selection.

Intensive Care Med, 1988, 14(6), 595 - 601
The effect of nifedipine alone or combined with low dose acetylsalicyclic acid on endotoxin-induced pulmonary hypertension in the piglet; Schranz D et al.; Cardiovascular responses to the calcium antagonist nifedipine, alone and combined with low dose acetylsalicyclic acid (ASA), were evaluated in a piglet model of endotoxin-induced pulmonary hypertension . All animals were anesthetized, paralyzed and mechanically ventilated . Cardiac output (CO), pulmonary artery pressure (PAP), aortic blood pressure (SAP), pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAPM) and arterial blood gases were measured before and after induction of pulmonary hypertension by E . coli endotoxin and after treatment . Results of treated groups were compared to a control group of piglets subjected to the same dose (0.15 micrograms/kg i.v.) of endotoxin . Control animals responded to a bolus injection of endotoxin within 15 min with an increase in mean PAP by 110% . Pulmonary vascular resistance (PVR) increased by 144% . Mean arterial pressure did not change significantly from baseline values . In animals treated with a single dose of 1 mg/kg ASA prior to endotoxin, the initial pulmonary response was not quantitatively different from control values, whereas ASA 20 mg/kg abolished the pulmonary vascular reaction . The increase of systemic vascular resistance (SVR) produced by endotoxin was aggravated by high dose ASA . In piglets treated with nifedipine (4 micrograms/kg/min) over 30 min after the application of endotoxin with and without additional infusion of nifedipine 60 min prior to endotoxin the PVR could be attenuated . The combination of nifedipine and low dose ASA showed synergistic effects compared to control . The increase of mean PAP was significantly reduced, the PVR remained in baseline range due to a marked elevation of cardiac output.

Complement, 1988, 5(3), 120 - 9
Enhancing effect of autologous human erythrocytes on generation of C3 cleavage products beyond iC3b; Jepsen HH et al.; The in vitro formation of C3d and C3c in fresh normal human serum (NHS) after addition of five different activators of the complement (C) system was studied . Following C-activation in NHS (n = 53) by Sephadex G-200 beads, the conversion of C3 was found to proceed to iC3b with a variable but restricted generation of C3d . Similar results were obtained by use of heat-aggregated IgG, Escherichia coli, zymosan, and cobra venom factor . However, comparing the C3d concentration following activation in the presence and absence of autologous red blood cells (RBC) at 37 degrees C the generated C3d was found to be 2- to 3-fold higher in the presence of RBC after 30, 60, and 210 min . Preincubation of RBC with polyclonal anti-CR1 antibodies resulted in a dose-dependent reduction of the amount of C3d generated . C-activation induced by Sephadex G-200 beads, in the absence of RBC, generated iC3b without a significant production of C3d . After removal of the activator beads, addition of RBC resulted in a decrease of iC3b and a clear increase in the C3c and C3d concentration within 3 h . Western blotting analysis of the C3d produced in the presence of RBC showed that the molecular weight (36 kilodaltons) was similar to that of C3d formed in vivo.

Cancer Immunol Immunother, 1988, 27(3), 272 - 7
Mitogenic, immunoadjuvancy, and genetic studies on fatty acyl maltose; Bissonnette E et al.; Three synthetic glycolipids, maltose tetrapalmitate (MTP), maltose hexastearate (MHS), and maltose hexalinoleate (MHL) prepared as nontoxic lipid A analogs, and Escherichia coli lipopolysaccharide (LPS) were assayed for their mitogenic activity using spleen lymphocytes in nine inbred mouse strains and three F1 hybrids . The MTP and LPS were also assayed for their ability to enhance plaque-forming cell (PFC) responses using sheep red blood cells as the antigen in the same inbred mouse strains and F1 hybrids, The mitogenic activity of synthetic glyco-lipids was several fold lower than that of LPS and MHL was inferior to MTP and MHS . DBA/2J was the most responsive strain for MTP and DBA/1J and C3H/HeJ the least . The mitogenic activity of MTP was generally in agreement with the PFC response stimulation by it . Low-dose cyclophosphamide treatment of mice synergized MTP for PFC response augmentation . Genetic studies on MTP mitogenicity revealed that 90% of responder DBA/2J X nonresponder C3H/HeJ F1 hybrids had intermediate mitogenic activity . Among F2, 73% had intermediate-high activity and 27% were nonmitogenic . Among F1 X C3H/HeJ backcrosses 11% had high, 56% intermediate, and 33% had no mitogenic activity, whereas, for the F1 X DBA/2J backcross, 14% had high, 36% intermediate, and 50% low or negligible activity . The data favored a single gene for MTP activation of immune cells.

Microbiol Immunol, 1988, 32(6), 635 - 9
Linkage of K99 production and STa activity in a plasmid of an Escherichia coli porcine isolate; Shimizu M et al.; An Escherichia coli strain ZP118 of porcine origin was shown to harbor a 68-megadalton (Md) plasmid coding for a colonization factor K99 and heat-stable enterotoxin (STa), and a self-transmissible 51-Md plasmid coding for drug resistance . One of the transconjugants obtained by mating between ZP118 and E . coli C was found to harbor a 90-Md plasmid coding for K99, STa and drug resistance . Restriction endonuclease analysis suggested that the 90-Md plasmid could be a recombinant between the 68-Md plasmid and the 51-Md plasmid.

J Basic Microbiol, 1988, 28(1-2), 119 - 28
The effects of some protein-modifying reagents on the interaction of colicins A, E2, E3, and K with their respective Escherichia coli cell receptors; Smarda J et al.; Colicins attach themselves--through specific protein-protein interactions--onto receptors in the outer membrane of sensitive bacterial cells . An attempt was made to analyze amino-acid groups and attractive forces involved in this interaction, following treatment of either colicins A, E2, E3 and K or sensitive bacteria with various physico-chemical factors and several protein-modifying reagents . The amounts of colicin bound were checked by a quantitative biological assay . Ionic conditions and specific spatial conformation of both colicin and its receptor molecules are crucial in their interaction and, hence, in the biological effect of colicin . Formaldehyde, naphthalene-diisocyanate and osmium tetroxide strongly inhibit the binding ability of all colicins tested . The results suggest that NH2 and SH groups are involved in their binding onto receptors; also, CH3S groups seem to be engaged in the attachment of colicins E2 and E3 and phenol-OH groups in that of colicin K . The possible involvement of further groups (NH, SH etc.) should be checked using more specific reagents . The attitude of colicins E2 and E3 to their common receptor Btu B protein is nearly, but not completely the same . Receptors for all colicins tested should be oxidized to achieve optimal interactions; obviously, carbonyl groups are produced and newly formed anions increase the negative load of bacterial surface . In agreement, reduction of at least colicins A and E3 enhances their receptor binding reactivity . The binding capacity of each receptor can be modulated by a set of amino acid reagents in a specific manner.

Int J Biochem, 1988, 20(8), 811 - 5
Stopped flow kinetic studies of adenosine triphosphate phosphoribosyl transferase, the first enzyme in the histidine biosynthesis of Escherichia coli; Dall-Larsen T; 1 . Stopped flow kinetic studies of ATP phosphoribosyl transferase (EC 2.4.2.17) from Escherichia coli showed that high protein concentration and elevated temperature do not give a drastic reduction of the transferase activity when measured before inhibitory amounts of PRibATP (product) has accumulated . 2 . A small and slow increase in activity follows a reduction of the protein concentration showing a slow dissociation of the enzyme from an inactive to an active species . 3 . By lowering the concentration of the inhibitor histidine, a fairly slow increase in activity is observed indicating a dissociation of the enzyme . 4 . AMP and histidine together give a strong inhibition of the activity, while AMP alone stimulates the enzyme activity.

Antonie Van Leeuwenhoek, 1988, 54(3), 229 - 33
Phage-receptor on the cell wall of Veillonella rodentium; Totsuka M; Veillonellophage N2 prevented from adsorbing to Veillonella rodentium ATCC 17743 cells treated with polymyxin B, and also to lipopolysaccharides (LPSs) of the host cells treated with antibiotics . Therefore, these results indicate that receptor to phage N2 is cell wall LPSs . The LPSs of V . rodentium ATCC 17743 cells as receptor were characterized . Lipid A and total carbohydrate accounted for approximately 40% of the weight of the lipopolysaccharide complex . Heptose and 2-keto-3-deoxyoctonate were also present . Amino compounds included glucosamine, galactosamine, and glycine.

Proteins, 1988, 3(4), 209 - 18
Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: application to the DNA binding domain of lac repressor from Escherichia coli; de Vlieg J et al.; The technique of two-dimensional nuclear magnetic resonance (2D-NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution . In order to generate spatial structures from the atom-atom distance information obtained by the NMR method, different procedures have been developed . Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies . We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 "hydrogen bond" distance constraints . Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints . Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.

Med Microbiol Immunol (Berl), 1988, 177(4), 219 - 28
Modulating action of Escherichia coli heat-stable enterotoxin (STa) on the humoral immune response; Zucato MR et al.; The effect of Escherichia coli enterotoxin STa on the primary and secondary immune response in F1 (CBA x C57 B1/10) mice immunized against sheep red blood cells (SRBC) was investigated . Modulating action on the IgM and IgG response was found to be dependent on the dose-time administration of the toxin . Immunosuppression of the primary response on the 4th day after immunization was observed when the toxin was injected 15 min before the SRBC, followed by immunostimulation on the 6th day after antigen (Ag) injection . Moreover, toxin administration 48 h before SRBC caused immunosuppression of the primary immune response on the 4th and 6th days . On the other hand, the IgM and IgG secondary immune response, determined 6 days after boosting, was greatly enhanced by toxin administration 15 min before priming (day 0) or boosting (day 26) and 48 h before priming . The same response was suppressed by toxin administration 48 h before booster antigen injection.

Med Microbiol Immunol (Berl), 1988, 177(4), 195 - 205
Frequent detection of antibodies to hepatitis B virus x-protein in acute, chronic and resolved infections; Hess J et al.; Recombinant MS2- or beta gal fusion proteins containing parts of hepatitis B virus (HBV) HBx-, HBc-, and HBs-amino acid sequences were expressed in Escherichia coli and were used to screen 96 and 60 serum samples of HBV infected and uninfected patients, respectively, for the corresponding antibodies by immunoblotting . Antibodies against HBx were detected in 20 out of 65 sera of patients with previous resolved HBV-infection, in 3 out of 7 patients with persistent infection, and in 9 out of 24 sera of patients with acute HBV infection . The specificity of the immune reaction was confirmed by competition experiments with MS2- and beta gal-HBx fusion proteins, and by the lack of HBx antibodies in the sera of uninfected patients . Hbs and HBc antibodies were detected less frequently by immunoblotting with recombinant fusion proteins than by a commercial immunoassay . Our results indicate that HBx antibodies are induced early and frequently during HBV infection suggesting that the HBx protein is an early antigenic protein expressed in vivo.

Bull Soc Pathol Exot Filiales, 1988, 81(2), 202 - 10
{Incidence of pathogenic Escherichia coli in acute infantile diarrhea in Dakar}; Aidara A et al.; We have studied the incidence of enteropathogenic (EPEC), enteroinvasive (EIEC) and enterotoxigenic (ETEC) Escherichia coli associated with infant acute diarrhoeal disease in Dakar during a period of one year . We report 405 strains of Escherichia coli suspected to be the etiologic agent of the diarrhoea and isolated from 405 diarrheic stools of 0-5 years old children . We have isolated 119 pathogenic Escherichia coli with 63 EPEC (15.5%), 3 ETEC (0.7%) and 53 ETEC (13.1%) including 23 strains releasing heat-labile enterotoxin (LT+) and 30 strains releasing heat-stable enterotoxin (ST+) . No ST+/LT+ strain was isolated . Escherichia coli with colonization factor antigens were isolated from 62 children . Almost all of them are CFAI+ . Only one strain is CFAII+ and another one agglutinates with both CFAI and CFAII antisera . Among these CFA+ strains 5 belong to the EPEC group, 29 are enterotoxigenic (25 ST+ and 4 LT+) and 28 do not belong to any known etiopathologic group . Near 70% of the pathogenic Escherichia coli are from infants less than one year old, with a highest frequency between 7 and 12 months . Prevalence of ETEC is higher during the raining season . The existence of a great number of strains that belong to none of the 3 groups of etiopathologic Escherichia coli emphasis the need to search other factors of pathogenicity.

Mikrobiologiia, 1988 Jan-Feb, 57(1), 65 - 72
{Changes in the energy status of synchronous Escherichia coli cultures during aerobic-anaerobic transitions}; Tkachenko AG et al.; The behaviour of energy and growth parameters for a synchronous Escherichia coli culture was studied in the course of aerobic-anaerobic transitions induced by either reversible or constant cessation of aeration at different phases of the cell cycle . The intracellular concentrations of ATP, ADP and AMP were assayed using a technique developed by the authors and based on thin-layer chromatography of adenyl nucleotide dansyl derivatives . Prolonged anaerobiosis led to profound alterations in the energy status which diminished the energy flux in constructional processes and preserved the energy resources for maintenance needs . The response of the energy parameters to a transitory anaerobic shock depended on the phase of synchronous growth and, apparently, on the ratio between the rates of reactions supplying and consuming the energy, which underwent changes in the cell cycle.

Physiol Behav, 1988, 42(3), 287 - 91
Effects of chronic infusion of lipopolysaccharide on food intake and body temperature of the rat; O'Reilly B et al.; Unrestrained male Sprague-Dawley rats were infused for seven days with a low (2.45 micrograms/hr) or high (9.81 micrograms/hr) concentration of E . coli lipopolysaccharide (LPS) . Compared to control (saline-infused) rats, food intake in the LPS-infused rats remained depressed for the entire infusion period . Despite this long-term suppression of food intake, fever was observed only during the daytime hours for the first two days of infusion . No significant increase in nighttime body temperature was observed . These data indicate that although tolerance to LPS occurred in rats with regard to its fever-inducing effect, tolerance with respect to its anorexigenic action did not occur.

Dermatologica, 1988, 176(6), 288 - 92
Cutaneous malakoplakia . Report of a case; Palou J et al.; A case of cutaneous malakoplakia in a 44-year-old women under immunosuppressive therapy after renal transplantation for nephrosclerosis is reported . A nodular lesion with inflammatory signs appeared on her right buttock . Histopathology showed a dermal infiltrate with histiocytes containing Michaelis-Gutmann bodies . Electron microscopy showed Michaelis-Gutmann bodies with concentric laminar structure ('target' appearance) . Tissue culture revealed Escherichia coli growth . Lesions recurred on surrounding skin and left buttock after excision . Clofazimine treatment resulted in total remission.

Eur Surg Res, 1988, 20(3), 211 - 9
Changes in granulocyte chemiluminescence and plasma fibronectin concentrations following major blunt trauma; Perttila J et al.; Chemiluminescence activity of granulocytes in phagocytosis of zymosan and Escherichia coli and their responses to chemoattractant N-formylmethionyl-leucyl-phenylalanine (FMLP) were evaluated in 13 major blunt trauma patients (Injury Severity Score 31 +/- 6) and their plasma fibronectin concentrations were measured . Chemiluminescence responses to zymosan and E . coli were at control levels immediately after injury and a week thereafter, but responses to FMLP were increased compared to the controls (p less than 0.05) . Plasma fibronectin concentrations were depressed on the day after trauma (p less than 0.001) but increased to control values over 1 week . The changes had no correlation with the patients' recovery.

Eur Surg Res, 1988, 20(3), 195 - 204
Indications of chlormethiazole as a protective agent in experimental endotoxemia; Modig J; Chlormethiazole, which is derived from the thiazole moiety of thiamine, possesses sedative, hypnotic and anticonvulsant properties . This anesthetic agent was compared with ketamine in a porcine model of endotoxemia to evaluate effects on cardiovascular and pulmonary function, oxygen delivery and survival . Continuous 6-hour intravenous infusion of Escherichia coli endotoxin caused pronounced pulmonary and cardiovascular derangement and decreases in oxygen delivery and pH in 13 pigs given ketamine anesthesia . Eight of thirteen pigs survived the observation period . Contrastingly, 10 pigs given chlormethiazole anesthesia and endotoxin showed a significantly attenuated response . Thus, the increases in mean pulmonary arterial pressure, venous admixture and extravascular lung water were significantly lower and the decreases in cardiac output, oxygen delivery and pH were significantly modified by chlormethiazole . All 10 pigs survived the observation period . Chlormethiazole may increase the clearance of endotoxin and thus ameliorate the endotoxin response . Although extrapolating from animal data requires great caution, these data may favor the use of chlormethiazole in septic states requiring surgical intervention and anesthesia and as sedation in critically ill septic patients in our intensive care units.

Ann Rech Vet, 1988, 19(1), 59 - 64
{Epidemiology of diarrhea caused by Escherichia coli and rotavirus in calves and lambs in Morocco}; Fassi-Fehri MM et al.; An epidemiological survey on E coli and rotavirus associated diarrheas in one to twenty five days old calves and lambs was made in three regions: Rabat-Kenitra, Marrakech and Agadir . Isolated E coli K99 stains have been studied of a biochemical, serotypical (O antigen) and antibiotypical point of view . The identification of rotavirus was made by ELISA test . Persistence of K99 antigen and heat stable toxin A was examined after a conservation of 5 weeks at - 18 degrees C . The frequency of E coli K99 or rotavirus associated diarrheas is respectively 26.9% and 29.7% in calf, 10% and 30% in lamb . This incidence considerably decreases from the 20th day in calf and from the 11th day in lamb . It must be observed that 34.8% of cases of diarrheas in calf and 55% in lamb cannot be ascribed to investigated agents . Only 12 out of 42 E coli K99 strains belong to serogroups O101, O8 and O9 . Preservation of strains to - 18 degrees C comes with the loss of K99 antigen . These strains are not toxinogens . Among the strains having kept this antigen, 29% are toxinogens . Surveyings of antibiotics resistance was discussed.

Proteins, 1988, 3(2), 102 - 12
Site-directed mutations altering methyl-accepting residues of a sensory transducer protein; Nowlin DM et al.; The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli . Transducers are methyl-accepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation . In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg . The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation . Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein . Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg . The mutant proteins were less efficient than normal Trg in mediating adaptation . Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.

Princess Takamatsu Symp, 1988, 19, 73 - 86
IL-2 receptor and Fc epsilon R2 gene activation in lymphocyte transformation: possible roles of ATL-derived factor; Yodoi J et al.; Lymphocyte transformation/activation is accompanied by the induction of a variety of the inducible receptors associated with the activation antigens . The p55 chain of the interleukin-2 receptor (IL-2R/p55) recognized by anti-Tac monoclonal antibody (mAb) is expressed on activated T and B cells as well as natural killer (NK) cells . IL-2R/p55(Tac) is constitutively expressed on T4(+) T cells transformed by human T-lymphotropic virus I (HTLV-I), which is a causative agent for adult T-cell leukemia (ATL) . Low affinity Fc receptor for IgE (Fc epsilon R2/CD23) is another inducible receptor binding IgE and is expressed on various hematopoietic cell types . While the physiological expression of Fc epsilon R2 and its soluble form (IgE Binding Factor; IgE BF) is variably regulated by cytokines and IgE, there is a constitutive expression of Fc epsilon R2 on Epstein-Barr virus (EBV) transformed B cell lines and some of HTLV-I (+) T cell lines . ATL-derived factor (ADF) has been characterized as an IL-2R/p55(Tac) inducing factor derived from HTLV-I(+) T cell lines . Purification of ADF protein and the cDNA cloning proved that ADF is closely related to the autocrine growth factor produced by an EBV(+) B cell line 3B6 (H . Wakasugi and T . Tursz) and belonging to the family of thiol reducing co-enzyme thioredoxin which is involved in many biological reactions . Recombinant ADF produced by COS cells and E . coli showed both IL-2R inducing and reducing activities . We will discuss the biological roles of ADF in viral and normal lymphocyte activation and transformation in relation to the receptor gene activation.

Arch Virol, 1988, 102(3-4), 245 - 62
Adenovirus transcriptional complexes contain EIa encoded tumour antigens physically bound to cellular proteins; Mirza A; Adenovirus type 12 transcriptional complexes were isolated from cells during the early phase of infection . Sedimentation analysis identified a fast sedimenting complex type I and a slow sedimenting complex type II . Both complexes made virus specific RNA complementary to all the early genes and both contained viral DNA, which in type II but not in type I had nucleosome like configuration . Analysis of the proteins of the complexes with antiserum against Ad 12 EIa-beta-galactosidase fusion protein expressed in E . coli demonstrated the following: (a) type I complex contained EIa 45 K protein, which co-precipitated with cellular proteins of mol . wt . 42, 58, and 60 K, (b) type II complex contained EIa 47 K protein, which co-precipitated with major cellular proteins of 35, 40-46 K and minor proteins of 58, 60, 68, 76, 86, and 120-150 K . Association of EIa specific and cellular proteins to transcriptional complexes was sensitive to both 1 M NaCl and DNAse I indicating the DNA binding nature of these proteins . Treatment of transcriptional complexes with 1 M NaCl or DNase I released EIa proteins, which still remained strongly bound to cellular proteins . These findings suggested that EIa proteins bind to viral DNA and that this binding is probably mediated by cellular proteins.

Vet Res Commun, 1988, 12(4-5), 305 - 11
Intestinal permeability to macromolecules during colibacillosis in piglets; Vellenga L et al.; Intestinal macromolecular permeability to macromolecules was determined in a group of specific pathogen free piglets before and after they were infected experimentally with Escherichia coli . Six hours after the infection all piglets developed a profuse diarrhoea . The urea and total protein concentrations in the serum increased markedly after the onset of diarrhoea . Haemoglobin concentration and PCV decreased steadily during the experiment but blood glucose concentration and lipid composition of the faeces did not change . No structural abnormalities in the jejunal and ileal mucosa were seen . No uptake of macromolecules (40,000 KDa) was found suggesting that molecules with a molecular weight of 40,000 or more do not play a role in the persistent diarrhoea sometimes seen in piglets after colibacillosis.

EMBO J, 1988 Jan, 7(1), 261 - 8
Three-dimensional reconstruction of maltoporin from electron microscopy and image processing; Lepault J et al.; Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis . Two different trimer packing forms were observed . One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm) . In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing . At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane . The pore defined by maltoporin has a similar structure to that outlined by the matrix protein . From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit . A tentative outline of the maltoporin promoter is given . Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.

Tokai J Exp Clin Med, 1988, 13 Suppl, 227 - 34
Molecular cloning and analysis of P . 69, a vir-controlled protein from Bordetella pertussis; Charles IG et al.; The cloning sequencing and analysis of an important antigenic component of Bordetella pertussis is described . The gene for P.69, in common with a variety of other so called "virulence" genes, (e.g., adenylate cyclase (AC), pertussis toxin (PT) and filamentous haemagglutinin (FHA)), is under control of the vir locus . The protein P.69 is externally localised on cells and protein preparations are protective as judged by the mouse intra-cerebral challenge test . The gene encoding the P.69 antigen was isolated by hybridization of mixed oligonucleotide probes against B . pertussis genomic DNA . These oligonucleotides were designed from the protein sequence data obtained from a cyanogen bromide digest of the P.69 protein . DNA sequence analysis reveals a G:C rich gene capable of encoding a protein of 910 amino acids and Mr of 93478, whose likely promoter and ribosome binding sites show little homology to their E.coli counterpart . In common with some of the genes in the PT operon the sequence 5'-CCTGG-3' was found 5' to the ATG initiation codon . At the 3', end 29 bases after the TAA stop codon, the sequence 5'GTTTTTCCT-3' was found in an equivalent position to the same sequence in the PT operon . Examination of the protein sequence reveals two regions with directly repeated elements (GGAVP)3(GGFGP)2 and (PQP)5.

Development, 1988, 104 Suppl, 75 - 83
The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences; Laughon A et al.; The ANT-C gene cluster is part of a network of genes that govern pattern formation in the development of Drosophila . The ANT-C genes encode proteins that contain a conserved 60 amino acid sequence, the homeodomain . Here we show that the homeodomains encoded by two of the ANT-C loci confer sequence-specific DNA-binding activity . The DNA sequence specificities of the Dfd and ftz homeodomains appear to overlap completely in vitro, indicating that differences in regulatory specificity among ANT-C and BX-C proteins (assuming that differences exist) must be a consequence of the nonconserved protein sequences found outside of the homeodomains . Deletions that remove sequences from either end of the ftz homeodomain abolish DNA-binding activity, consistent with the commonly held assumption that the homeodomain is a structural domain . The relevance of in vitro DNA-binding experiments to the regulatory function of ftz is supported by our finding that a temperature-sensitive ftz mutation that causes a pairwise fusion of embryonic segments also reduces the affinity of the ftz homeodomain for DNA . Restriction fragments containing ftz homeodomain binding sites were identified within a 90 kb stretch of DNA extending the Antp P1 and P2 promoters . Binding sites appear to be clustered near the P1 promoter but also occur near P2 and in the region between the two . The task remains of determining which of these sequences mediate regulation of Antp by ftz or by other genes that encode closely related homeodomains.

Free Radic Biol Med, 1988, 5(4), 215 - 23
Degradation of oxidatively denatured proteins in Escherichia coli; Davies KJ et al.; When exposed to oxidative stress, by oxygen radicals or H2O2, E . coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation . The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen . Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically . Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation . Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis . Two-dimensional polyacrylamide gel electrophoresis of {3H}leucine labeled E . coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected . To test for the presence of soluble proteases, we prepared cell-free extracts of E . coli and incubated them with radio-labeled protein substrates . E . coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase . When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E . coli extracts . Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility . Pretreatment of E . coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunopharmacol Immunotoxicol, 1988, 10(4), 501 - 12
Differential effect of mixed D1/D2 and selective D2 dopaminergic antagonists on mouse T and B lymphocyte proliferation and interleukin production in vitro; Boukhris W et al.; The effects of some dopaminergic antagonists were investigated on mouse lymphocyte proliferative responses in vitro . The mixed D1/D2 dopaminergic antagonists chlorpromazine, haloperidol and flupentixol inhibited 3H-Thymidine incorporation into adult BALB/c mouse spleen cells stimulated by concanavalin A, lipopolysaccharide from Escherichia coli, and allogenic cells in a mixed lymphocyte reaction . The inhibition was achieved at concentrations greater than 10(-6) M . It was not accounted for by decreased cell viability and it was no longer demonstrable when the compound was added 24 h or 48 h after the mitogenic stimulus . Conversely selective D2 dopaminergic antagonists sulpiride, metoclopramide and domperidone had no inhibitory effect at concentrations ranging from 10(-9) to 10(-5) or 10(-4) M . The three mixed D1/D2 antagonists inhibited the mitogenic effect of interleukin-1 on concanavalin A-stimulated thymocytes, but not the activity of interleukin-2 on the proliferation of the CTLL-2 cell line . The mixed D1/D2 antagonists interfered with the production of interleukin-2 but not with that of interleukin-1 . These results indicate that dopaminergic antagonists may differentially affect lymphocyte proliferative responses to T or B cell mitogens or alloantigens . The mechanisms involved in terms of receptor specific or non specific phenomenons are discussed.

Nucleic Acids Symp Ser, 1988, (19), 175 - 8
Studies on essential amino acid residues and functional regions of H+-ATPase (F0F1) from Escherichia coli by gene manipulation; Noumi T et al.; We discussed application of in vitro mutagenesis on H+-ATPase (F0F1) of Escherichia coli . The oligonucleotide-directed site specific mutagenesis and construction of a set of truncated subunits were useful for identifying essential residues of beta subunit and a functional region of epsilon subunit, respectively, of this complicated membrane enzyme.

J Basic Microbiol, 1988, 28(6), 381 - 4
Identification by heteroduplex analysis of an invertible element (Min) common among IncM group plasmids; Karste G et al.; The analysis of heteroduplexes between EcoRI digested DNA derived from IncM plasmids in the transfer-off and in the transfer-on state revealed an internal loop of about 1 kbp on a distinct large EcoRI fragment . The presence of an invertible element (Min) is suggested which enables the formation of conjugative pili at 30 degrees C, but switches off the pilus formation at 37 degrees C incubation temperature.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1988, (8), 89 - 93
{The action of sodium and potassium ions on the H+-ATPase activity of Escherichia coli K12 (lambda) membranes}; Nerkararian AV; The dependence of activity of H+-ATPase membranes of Escherichia coli K12 (lambda) grown anaerobically of potassium and sodium ions has been studied . The addition of K+ or Na+ to the reaction mixture causes an increase of H+-ATPase activity . The effect depended on conditions and keeping time of the preparation of membranes . The sensitivity of enzyme to potassium and sodium decreased with the rise of temperature from -20 degrees C to -4 degrees C and an increase of keeping time.

Antonie Van Leeuwenhoek, 1988, 54(4), 285 - 99
Genetic organization and biogenesis of adhesive fimbriae of Escherichia coli; Oudega B et al.; The genetic organization of the determinants of type 1, K88ab, K99 fimbriae and P(pap)pili of Escherichia coli is presented . The functions of the various gene products are described and a model for the process of fimbriae biogenesis is presented and discussed.

Avian Dis, 1988 Jan-Mar, 32(1), 74 - 8
Adhesin-receptor interactions mediating the attachment of pathogenic Escherichia coli to chicken tracheal epithelium; Gyimah JE et al.; Specific adherence of pathogenic Escherichia coli (serotypes O1, O2, and O78) to chicken tracheal epithelium was investigated using adherence-inhibition procedures . The role of pilus as adhesin was studied by blocking the pilus with antipilus antibodies . The nature of the host cell receptor was determined by blocking bacterial adhesion with specific carbohydrates or lectins and destroying the receptor with sodium metaperiodate . Antipilus antibodies to all three serotypes significantly (P less than or equal to 0.05) inhibited their adherence . Sodium metaperiodate considerably inhibited the adhesion of all three serotypes, indicating a role for monosaccharides in the host cell receptor . D-Mannose and its derivative methyl-alpha-D-mannopyranoside inhibited the adhesion of serotypes O1 and O78, indicating a role for these sugars in the host cell receptor; this was further supported by the inhibition of both serotypes after treatment of tracheal epithelium with concanavalin A . None of the sugars or lectins used inhibited adhesion of serotype O2.

Mol Microbiol, 1988 Jan, 2(1), 73 - 80
Biogenesis of F7(1) and F7(2) fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein; Riegman N et al.; The F7(1) and F7(2) P-fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985) . With the immunocytochemical gold-labelling technique it was demonstrated that both the FsoE and FstE proteins are non-adhesive minor fimbrial subunits located at the tip of the fimbrial structure . The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.

Gynecol Obstet Invest, 1988, 25(1), 31 - 4
Antibodies against enterotoxigenic Escherichia coli in the colostrum isolated from infants with diarrhea; Kletter Y et al.; The role of certain types of Escherichia coli in infectious diarrhea in infants and young children is well known . Infants who are breast-fed are less prone to gastroenteritis during their first year of life . Antibodies against three types of fimbrial antigens (adhesions) of enterotoxigenic E . coli (ETEC) in the colostrum were studied . The hemagglutination inhibition test method was used to detect antibodies against ETEC adhesions, i.e . colonization factors, I and II and fimbria type I . The colostrum of mothers on the 1st and 3rd day post partum was standard for the presence of antibodies . The results show that most of the colostrum samples contained antibodies against adhesions of the types of ETEC that we worked with . This study will enhance the knowledge as to why mothers should breast-feed their babies.

J Bacteriol, 1988 Jan, 170(1), 459 - 62
Use of lac fusions to measure in vivo regulation of expression of Escherichia coli proton-translocating ATPase (unc) genes; Angov E et al.; In-frame fusions to lacZ were constructed in two adjacent genes of the unc operon of Escherichia coli, uncA and uncG, which code for the alpha and gamma subunits of the proton-translocating ATPase . After each fusion was moved into the E . coli chromosome, measurement of beta-galactosidase activities from single-copy genes showed that uncA was expressed significantly better in vivo than was uncG, but the relative expression dependent on the chromosomal location of each fusion and the presence or absence of other unc genes.

J Bacteriol, 1988 Jan, 170(1), 179 - 83
Temperature-sensitive Escherichia coli mutant with an altered initiation codon of the uncG gene for the H+-ATPase gamma subunit; Miki J et al.; A mutant of Escherichia coli showing temperature-sensitive growth on succinate was isolated, and its mutation in the initiation codon (ATG to ATA) of the uncG gene (coding for the gamma subunit of H+-ATPase F0F1) was identified . This strain could grow on succinate as the sole carbon source at 25 and 30 degrees C, but not at 37 or 42 degrees C . When this strain was grown at 25 degrees C on succinate or glycerol, its membranes had about 15% of the ATPase activity of wild-type membranes, whereas when it was grown at 42 degrees C, its membranes had about 2% of the wild-type ATPase activity . Membranes of the mutant grown at 25 or 42 degrees C could bind F1 functionally, resulting in about 40% of the specific activity of wild-type membranes . The gamma subunit was identified in an EDTA extract of membranes of the mutant grown at 25 degrees C, but was barely detectable in the same amount of extract from the mutant grown at 42 degrees C . These results indicate that initiation of protein synthesis from the AUA codon is temperature sensitive and that the gamma subunit is essential for assembly of F1 in vivo as shown by in vitro reconstitution experiments (S . D . Dunn and M . Futai, J . Biol . Chem . 255:113-118, 1980).

Infect Immun, 1988 Jan, 56(1), 241 - 6
Virulence properties of enterotoxigenic Escherichia coli O8: KX105 strains isolated from diarrheic piglets; Broes A et al.; Twenty-two enterotoxigenic Escherichia coli (ETEC) O8:KX105 strains isolated from 1- to 7-week-old diarrheic piglets were examined for virulence properties . Thirteen strains caused acute watery diarrhea in orally infected, colostrum-deprived newborn piglets, whereas the remaining nine did not . The enteropathogenic strains colonized the small intestine, albeit with lower intensity than classical porcine ETEC . They produced the heat-stable STb and heat-labile LT enterotoxins, whereas the nonenteropathogenic strains produced the STb enterotoxin alone . None of the E . coli O8:KX105 strains exhibited mannose-resistant hemagglutination with erythrocytes from 12 species . Ten of the enteropathogenic and two of the nonenteropathogenic strains were positive for mannose-sensitive hemagglutination . These strains produced rodlike fimbriae 3 to 5 nm in diameter, whereas no fimbriae were detected on the other strains . None of the 22 strains produced the fimbrial antigens F4, F5, F41, F2, F3, FY(Att 25), and F165 . Of the 13 enteropathogenic strains, 10 expressed the F6 antigen in the intestines of infected piglets but not in cultures . The other three enteropathogenic strains apparently lacked all of the known fimbrial antigens from porcine ETEC.

Infect Immun, 1988 Jan, 56(1), 1 - 6
Maternal immunization with P fimbriae for the prevention of neonatal pyelonephritis; Kaack MB et al.; Rhesus monkeys (Macaca mulatta) were immunized with purified P fimbriae from Escherichia coli during the last trimester of pregnancy . Infants born of these mothers were compared with those from nonimmunized rhesus mothers . A delay in the onset of renal disease after bladder infection showed protection from passive immunization . This was associated with a high antibody titer in serum . In addition to delayed onset of renal infection, a decreased number of immunized monkeys developed pyelonephritis.

Biol Met, 1988, 1(1), 62 - 8
Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12; Wee S et al.; The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid . The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose . The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue . All four cysteines were shown to be present as thiols . Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis . Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M . A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.

Microbiol Immunol, 1988, 32(12), 1221 - 34
Ecological studies on reovirus pollution of rivers in Toyama Prefecture; Matsuura K et al.; Reoviruses (reos) were isolated from river water in various areas of Toyama Prefecture . The frequency of reo isolation was higher in the river water, the basin of which has a larger human population . The degree of river contamination with reo paralleled that with the Escherichia coli group of organisms, and reos were frequently isolated from sewage, too . The high antibody-positive (greater than or equal to 1:8 or greater than or equal to 1:10) rates against reos in humans and other animals tested (swine, cattle, and field rodents) indicated their wide-spread infection with reos . These results suggested that the major source of reos present in the river water may be the excretion by humans and other animals, especially the former . Survival experiments in which reos were added into the filtered or centrifuged river water and kept at various temperatures, revealed that reos survived for more than 3 years at 5 C . In the field experiment where reos suspended in cellophane tubes were kept in an agricultural water stream in winter (water temperatures below 10 C), they survived for 6 months until the water temperature rose above 20 C in summer.

Nucleic Acids Symp Ser, 1988, (19), 39 - 42
Identification by a random linker insertion method of a region which suppresses the transforming activity of the raf oncogene; Ishikawa F et al.; Activation mechanism of raf oncogene was studied by applying in vitro mutagenesis to its cDNA . Previous studies suggested the presence of an activation suppressing sequence in the amino-terminal half of c-raf product . Loss of the sequence by genetic rearrangement was presumed to convert c-raf to possess transforming activity . To identify such sequence, we prepared cDNA mutants by random linker insertion . Synthetic oligonucleotide linker was inserted into the plasmid containing cDNA at a single and random site . Coupling two different mutants, in-frame deletion mutants were constructed systematically . Analysis of these deletion mutants revealed a region, the loss of which made c-raf activated.

Nucleic Acids Symp Ser, 1988, (19), 171 - 3
Construction of a vector for the study of regulatory mechanism of gene expression and its utilization in the melibiose operon of Escherichia coli; Shimamoto T et al.; We constructed a vector to evaluate terminator or attenuator of transcription quantitatively . This vector is a plasmid possessing lac promoter-polylinker-CAT(chloramphenicol acetyltransferase) gene . DNA fragment of interest can be inserted into the polylinker site, and the effect of the DNA fragment on the expression of the downstream gene is evaluated from the CAT activity . We analyzed gene expression of the melibiose operon of Escherichia coli using this plasmid, and found that a DNA fragment containing a large stem-loop structure with boxA sequence in it, which was present between melA and melB, caused strong reduction of gene expression . This DNA portion seems to be responsible for reduced expression of melB, the second gene of the melibiose operon.

Arch Virol, 1988, 103(3-4), 267 - 74
Immunological properties of an N-terminal fragment of herpes simplex virus type 1 glycoprotein D expressed in Escherichia coli; Kocken CH et al.; The N-terminal fragment, comprising residues -5 to 55 of herpes simplex virus type 1 glycoprotein D was expressed as a beta-galactosidase fusion protein in Escherichia coli . This gD-fusion protein reacts with monoclonal antibody LP 14 directed against glycoprotein D of HSV . Antisera obtained after immunization of rabbits with purified gD-fusion protein react with HSV-1 gD in a Western blot and with N-terminal synthetic peptides of gD . In addition, these antisera are able to neutralize viral infectivity in vitro.

Proteins, 1988, 4(1), 71 - 5
Crystallization and preliminary investigation of single crystals of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli; Cedergren-Zeppezauer ES et al.; Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme in the nucleotide metabolism that is a pyrophosphatase hydrolyzing dUTP, has been crystallized . The crystals belong to the trigonal space group R3 and diffract beyond 2 A . The native dUTPase crystals and a mercury derivative are stable in the X-ray beam and are suitable for a high resolution X-ray structure analysis.

Basic Life Sci, 1988, 47, 317 - 23
Activation of silent transposable elements; Burr B et al.; It is well known among maize geneticists that agents that cause chromosome breakage can activate quiescent transposable elements . However, other than temporarily relieving position effect, it is difficult to understand how these events can lead directly to activation . One possibility is that chromosome breakage can initiate a process in the cell resulting in a higher rate of spontaneous mutation . Such a system could be analogous to the SOS response of Escherichia coli in which an error-prone repair system is induced . Chemical mutagens that cause little chromosome breakage but add bulky adducts to the DNA can induce the SOS response . In seed homozygous for a1-m2(8004), wx-m8, no active Spm, that had been treated with ethyl methanesulfonate, we observed activation of Spm at the rate of 1.1 x 10(-4) . The spontaneous rate of activation in this material was 1.2 x 10(-5) . Most of the activation events occurred as single kernels . This result contrasts with sectors covering at least one-eighth of the ear that would have been expected if activation had occurred as a direct result of mutagenesis in the mature kernel . The late timing of these events suggests that the activation, in most instances, may not be the direct result of chemical mutagenesis.

Arch Virol, 1988, 101(1-2), 137 - 40
An improved procedure for the rapid one-step-cloning of full-length viroid cDNA; Puchta H et al.; The efficiency of viroid cloning can be increased by three to four orders of magnitude when the synthesis of viroid cDNA is primed in such a way that it carries identical sticky ends on both termini and when the multi-ion transformation is applied.

J Mol Evol, 1988, 27(2), 102 - 8
Structure of the human hemopexin gene and evidence for intron-mediated evolution; Altruda F et al.; The human hemopexin gene was isolated and its structure determined . The gene spans approximately 12 kb and is interrupted by nine introns . When the intron/exon pattern was examined with respect to the polypeptide segments they encode, a direct correspondence between exons and the 10 repeating units in the protein was observed . The introns are not randomly placed; they fall in the middle of the region of amino acid sequence homology in strikingly similar locations in 6 of the 10 units and in a symmetrical position in the two halves of the coding sequence . These features strongly support the hypothesis that the gene evolved through intron-mediated duplications of a primordial sequence to a five-exon cluster . A more recent gene duplication led to the present-day gene organization.

Allerg Immunol (Leipz), 1988, 34(1), 49 - 51
{Increased measuring rate of an in vivo phagocytosis test by using frozen samples}; Henke A; The in vivo phagocytosis test, as described by Dunn et al., allows only a limited number of measurements because the preparations do not last long . That's why it was tested, whether deepfreezing allows the evaluation of the preparations at a later time . The results show, that there were no differences between the counting of phagocytosed cells in fresh and in deep frozen macrophages, so that the experiments can be rationalized importantly by using the freezing technique.

Plasmid, 1988 Jan, 19(1), 68 - 70
Isolation and restriction endonuclease analysis of a mycoplasma plasmid; Bergemann AD et al.; A 1.7-kb plasmid was isolated from a large colony type strain of Mycoplasma mycoides subsp . mycoides . It was cloned into the Escherichia coli vector M13mp19, and DNA from the resultant recombinant was used to construct a restriction map of the plasmid . Because mycoplasmas show deviation from normal coding patterns, the availability of a mycoplasma plasmid could be useful in the development of a cloning vector for them.

Plasmid, 1988 Jan, 19(1), 46 - 56
Nucleotide sequence and copy control function of the extension of the incI region (incI-b) of Rts 1; Nozue H et al.; An Rts1 derivative, pTW20, contains three incompatibility (inc) regions, incI-a (incI in previous studies), incII, and newly determined incI-b loci . By restriction analysis, we have located the incI-b adjacent to the incI-a region on the pTW20 map . Nucleotide sequence analysis of the minimal incI-b region revealed the presence of four repeated sequences, each consisting of 18 bp, which is similar to the incI-a and incII repeats existing on mini-Rts1 . All four repeating units were required for expression of a strong incompatibility . In addition, RepA protein, essential for the replication of Rts1, bound specifically to the repeated sequences, suggesting that the repeats would titrate out RepA protein as do incI-a and IncII . Insertion of the incI-b to a mini-Rts1 plasmid in a natural arrangement decreases the copy number of mini-Rts1 to the same level as that of mini-F . The incI-a and incI-b might be a single constituent in incompatibility and copy number control of Rts1.

Proteins, 1988, 3(2), 121 - 9
Crystallization of purified recombinant human interleukin-1 beta; Carter DB et al.; The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +) . Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose . The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons . Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation . The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution . A structure determination based on these crystals is under way.

Biomed Biochim Acta, 1988, 47(1), K1 - 5
Abolition of DNA recognition site resistance to the restriction endonuclease EcoRII; Kruger DH et al.; The EcoRII recognition sites 5'-CCATGG-3' which occur three times in T3 DNA are refractory to this restriction endonuclease . However, these sites are specifically cleaved by EcoRII when a second, susceptible DNA species (pBR 322, phage lambda) is present.

Ann Inst Pasteur Microbiol, 1988 Jan-Feb, 139(1), 79 - 103
Variation of beta-galactosidase expression from Mudlac elements during the development of Escherichia coli colonies; Shapiro JA et al.; Bacterial colonies reveal the action of systems for multicellular regulation of genetic activity during growth and development . Colonies produced on agar indicator medium by Escherichia coli strains carrying the transposable genetic fusion element Mudlac displayed organized patterns of differential beta-galactosidase expression . One feature of these patterns was the presence of phenotypically distinct concentric rings containing cells that were not genetically distinct . A second feature of the patterns was the appearance of sectorial populations with novel phenotypes, frequently displaying coincident variation in several characters, such as enzyme activity, multicellular aggregation and rate of spread over the agar substrate . Subcloning analysis of sectors with more expansive growth phenotypes revealed that they contained bacteria expressing novel developmental sequences on more than one kind of medium . These novel developmental sequences could be transmitted to progeny bacteria but were reversible during growth in liquid medium . More stable clonal variation in patterns of beta-galactosidase expression arose during storage in liquid medium . Most of these changes correlated with transpositions or rearrangements of Mudlac sequences . Some changes in beta-galactosidase expression involved interactions between Mudlac elements and unlinked Mu derivatives . These results revealed the operation of novel control systems regulating beta-galactosidase expression from the lacZ sequences in a chromosomal Mudlac element and demonstrated at least two different kinds of clonal variation events which affected pattern formation in bacterial colonies.

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 270 - 6
{Alleviation of type I restriction in the presence of plasmids of group inc I . General characteristics and molecular cloning of the ard gene}; Kotova VIu et al.; The capability of a number of plasmids of incN and incI groups to alleviate an action of type I EcoK, EcoB, EcoD, and EcoA restriction endonucleases on the unmodified DNA was revealed . The efficiency of EcoK action on lambda 0 DNA is alleviated about 10 divided by 100 fold in E . coli K12 AB 1157 bacteria containing the plasmid of incN group (pKM101, N3, pJA4733) or incI group (R144, R648; R621a; ColIb-P9) . We have cloned ard gene of ColIb-P9 plasmid (SalI-C fragment) in pBR322 multicopying vector . A hybrid clone abolishing the EcoK restriction has been received . Ard gene activity is independent of the recA, recBc, recF, lexA, umuC, lon bacterial genes activity . Ard gene's product does not inhibit the EcoK restriction endonuclease action as well as ocr protein (phage T7) and does not increase the process of methylation of DNA as well as ral protein of phage lambda.

Gene, 1988, 62(1), 55 - 64
A plasmid vector system for the expression of a triprotein consisting of beta-galactosidase, a collagenase recognition site and a foreign gene product; Scholtissek S et al.; A plasmid-cloning vector system has been constructed which allows the production of fusion proteins with beta-galactosidase at the N terminus, followed by a recognition sequence for the site-specific protease, collagenase, and the foreign protein at the C terminus . A multicloning site allows the insertion of foreign genes in any translational reading frame . Fusion proteins were isolated by affinity chromatography on APTG-Sepharose . The foreign protein was released from the fusion product by collagenase cleavage . The vector was successfully utilized for the production of Escherichia coli single-stranded (ss) DNA-binding protein (SSB protein) . The proteolytically released SSB protein resisted elution from an ss DNA-cellulose column with 1 M NaCl.

Mol Microbiol, 1988 Jan, 2(1), 109 - 19
Identification and sequencing of the Escherichia coli cet gene which codes for an inner membrane protein, mutation of which causes tolerance to colicin E2; Drury LS et al.; Dominant mutations of the cet gene of Escherichia coli result in tolerance to colicin E2 and increased amounts of an inner membrane protein with an Mr of 42,000 . We have cloned the cet+ gene and sequenced its DNA, revealing that the gene product, coded by the longest open-reading frame, has an Mr of 49,772, with five predicted transmembrane structures towards its carboxy terminus and one at ist amino terminus . We have demonstrated that the cet locus does in fact code for the inner membrane protein that is present in increased amounts in cet mutants, and we have shown that this increased amount of Cet protein is the result of enhanced transcription . The cet gene is shown to be in the same operon as the phoM gene, which is required in a phoR background for expression of the structural gene for alkaline phosphatase, phoA . Although the Cet protein is not required for phoA expression, our experiments suggest that the Cet protein has an enhancing effect on the transcription of phoA . No effect of phosphate concentration on cet or phoM gene expression could be found and thus their primary function may not be connected to the phosphate regulon.

Mol Microbiol, 1988 Jan, 2(1), 1 - 8
Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400; Schmid K et al.; The scr genes located on plasmid pUR400 and responsible for sucrose (Scr) metabolism of Escherichia coli K12 and other enteric bacteria have been cloned on a 9.3 kb DNA fragment . The different genes were mapped by transposon insertion mutagenesis, by restriction endonuclease and deletion mapping, and the corresponding gene products were identified . Besides the known structural genes scrA, coding for an EnzymeII(Scr) (45 kD) of the phosphoenolypyruvate-dependent phosphotransferase system (PTS), and scrB, coding for a sucrose 6-phosphate hydrolase (invertase) (55 kD), two new structural genes were discovered . Gene scrK apparently codes for an intracellular and ATP-dependent fructokinase (39 kD), while scrY seems to code for a sucrose porin (58 kD) in the outer cell membrane . No genes for an Enzyme III(Scr) of the PTS or for (a) glycosyltransferase(s) were detected . The four genes form an scr operon (gene order, scrK scrY scrA scrB, transcription from K to B), regulated by a repressor (gene scrR, 37 kD) and inducible by sucrose, fructose and fructose-containing oligosaccharides.

Gene, 1988, 62(2), 301 - 13
The inducible lac operator-repressor system is functional for control of expression of injected DNA in Xenopus oocytes; Hu MC et al.; We have investigated the use of the Escherichia coli lac operator-repressor system to regulate the expression of genes introduced by microinjection into Xenopus laevis oocytes . We observe that expression of an MSV-cat fusion gene, in which the lac operator was inserted between the TATA box and the transcription start point (tsp), or between the tsp and the start codon (ATG), is completely repressed when the lac repressor protein is added to the plasmid suspension prior to injection . The lac repressor had no detectable effect on the expression of a coinjected HSV-1 tk gene that had no operator insertion (or on an MSV-cat gene without an operator), indicating that the nonspecific DNA-binding properties of the repressor do not inhibit transcription . CAT activity expressed from the operator-containing MSV-cat genes transcribed in the oocyte nucleus was also inhibited by repressor injected into the oocyte cytoplasm, showing that biologically active repressor proteins can enter the nucleus from the cytoplasm . Injection of the inducer IPTG into the oocyte cytoplasm markedly derepressed the repressed cat genes but not the HSV-1 tk gene coinjected as an internal control . Overall, our results show that the lac operator-repressor system can be useful as a genetic switch in the regulation of gene expression of injected DNA in frog oocytes . Finally, our observations on the vectors used in this work show that the MSV enhancer significantly activates transcription from the SV40 early promoter in frog oocytes, although previous studies have indicated that the MSV enhancer is not necessary for the activity of the MSV promoter in oocytes {Graves et al., Mol . Cell . Biol . 5 (1985) 1945-1958}.

Arch Microbiol, 1988 Jan, 149(3), 240 - 4
Two-dimensional gel electrophoretic analysis of Escherichia coli proteins: influence of various anaerobic growth conditions and the fnr gene product on cellular protein composition; Sawers RG et al.; Two-dimensional gel electrophoresis was used to examine the response of the cellular proteins of Escherichia coli to various anaerobic growth conditions and to the presence or absence of a functional Fnr protein . The steady-state levels of 125 polypeptides were found to vary in either a positive or negative manner, with many polypeptides being affected under a number of conditions . A large number (21) of the anaerobically inducible polypeptides were shown to be totally independent of the presence of Fnr while 22 were shown to be reduced in a fnr mutant under all anaerobic growth conditions tested . A total of 8 proteins were shown to be reduced in a fnr mutant only in aerobically grown cells indicating that the Fnr protein has a function in the presence of oxygen . This was further confirmed by the observation that 15 anaerobically inducible polypeptides were also found to show an increase in aerobically grown cells, however, only in a fnr strain . This latter finding implies that Fnr may also exhibit repressor function . This effect of Fnr-dependent repression was also observed with several polypeptides in anaerobically grown cells.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1988, (1), 86 - 90
{Permeability of Escherichia coli K-12 cells to exogenous nucleodepolymerases}; Egorov SIu et al.; The permeability of Escherichia coli cells for exogenous nucleodepolymerases has been studied by an immunoenzyme method . The enzyme ability to penetrate through the bacterial outer membrane and cell wall after 20 min of incubation with culture cells of delayed growth phase has been found.

Genes Dev, 1988 Jan, 2(1), 93 - 105
Identification and intracellular localization of the unc-22 gene product of Caenorhabditis elegans; Moerman DG et al.; The unc-22 gene is one of a set of genes identified using classical genetics that affect muscle structure and function in the free-living nematode Caenorhabditis elegans . Since cloning the unc-22 gene by transposon tagging, we have used conventional techniques combined with a set of Tc1 transposon insertion alleles to characterize the gene and its products . The gene extends over more than 20 kb of genomic sequence and produces a transcript of approximately 14 kb . A polyclonal antibody raised against an Escherichia coli beta-galactosidase-unc-22 fusion protein recognizes a polypeptide in nematode extracts that is between 500,000 and 600,000 daltons and labels the muscle A-band in indirect immunofluorescent microscopy . The Tc1-induced alleles have been used at every stage to verify these conclusions . The Tc1 insertions are spread over much of the region that contributes to the mature transcript; in most alleles, Tc1 sequences are incorporated into a composite unc-22-Tc1 transcript . The large protein is either absent or severely reduced in amounts in the mutants . In one case, a truncated polypeptide was also identified . The location of the protein in the A-band, along with earlier genetic data, suggests that the unc-22 product may interact with myosin to regulate its function.

Genes Dev, 1988 Jan, 2(1), 54 - 67
Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins; Hirochika H et al.; The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame . We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11) . Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components . The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA . The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses . Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1) . The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted . We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro . Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number . These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand . The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses . Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.

Radiobiologiia, 1988 Jan-Feb, 28(1), 7 - 10
{Use of the RI plasmid to study radiation damage to the methyl anti-nuclease barrier in the DNA of Escherichia coli}; Torosian MV et al.; Ionizing radiation (gamma-rays, 100 to 750 Gy) does not impair methyl anti-nuclease barrier in DNA of exposed cells . From this it follows that the methyl anti-nuclease barrier is radioresistant . It is assumed that restrictases are involved in degradation of single-strand DNA regions, that is, in degradation of replicative forks.

Mol Gen Genet, 1988 Jan, 211(1), 78 - 87
Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli; Old IG et al.; We have cloned the genes for the two homocysteine transmethylases of Escherichia coli K12 . The vitamin B12-independent enzyme is encoded by the metE gene while the metH gene codes for the vitamin B12-requiring enzyme . Overexpression of the gene products and Tn1000 mutagenesis have enabled the metE and metH gene products to be identified as 99 kDa and 130 kDa polypeptides, respectively . The truncated polypeptides generated by Tn1000 insertion were used to determine the direction of transcription of the metE and metH genes . Negative complementation suggests that the MetH enzyme exists as an oligomer . Investigation of the expression of the chromosomal- and plasmid-encoded gene products confirms that metE is subject to negative control by vitamin B12 and methionine, and that metH is under positive control by the cofactor and negative control by methionine . For vitamin B12 and methionine to act as regulatory effectors in metE control, functional metH and metJ genes are required, respectively . The use of stable Tn1000-generated fragments of the metE product as electrophoretic markers for the plasmid-encoded metE gene product demonstrated that the two regulatory proteins involved in negative control of metE are present in excess . Under conditions whereby both forms of negative metE control are non-functional, the metE gene product represented about 90% of the total protein, and cell growth was severely impaired.

Mol Gen Genet, 1988 Jan, 211(1), 176 - 82
Sequence, expression and localization of the immunity protein for colicin B; Schramm E et al.; Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane . The nucleotide sequence of the cbi region was determined . It contains an open reading frame for a polypeptide consisting of 175 amino acids . The amino acid sequence is homologous to the primary structure of the colicin A immunity protein . This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins . The immunity protein could be identified following strong overexpression of cbi . The electrophoretically determined molecular weight of 20,000 was close to the calculated molecular weight of 20,185 . The protein contains four large hydrophobic regions . The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane . It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.

Mol Gen Genet, 1988 Jan, 211(1), 138 - 42
Chromosome replication in Escherichia coli induced by oversupply of DnaA; Xu YC et al.; Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42 degrees C causes initiation of one extra round of replication in a dnaA+ strain grown in glycerol minimal medium . This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.

Mol Gen Genet, 1988 Jan, 211(1), 106 - 12
Expression of the Escherichia coli dnaQ (mutD) gene is inducible; Quinones A et al.; By promoter fusion to the galK gene and comparative S1 analysis we investigated the in vivo regulation of transcription of the dnaQ gene which encodes the epsilon-subunit of the DNA polymerase III holoenzyme carrying the 3'----5' exonucleolytic proofreading function . Induction of a mutagenic stress situation by treatment with the base analogue 2-aminopurine (2-AP) leads to an increase in dnaQ transcription . S1 mapping analysis of the two dnaQ transcripts revealed a differential promoter activation for this 2-AP induced increase in dnaQ transcription . In addition, a similar galK promoter fusion with the dnaN gene coding for the beta-subunit of the DNA polymerase III holoenzyme revealed that dnaN transcription is also 2-AP inducible as judged by galactokinase activity . This is the first evidence for the inducibility of dnaQ gene expression (and possibly of other genes of the DNA polymerase II holoenzyme) and is discussed in relation to DNA repair mechanisms.

Mol Gen Genet, 1988 Jan, 211(1), 1 - 7
Quinolone-resistant mutations of the gyrA gene of Escherichia coli; Yoshida H et al.; DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones . The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E . coli chromosome . It encoded a polypeptide of 875 amino acids with a molecular weight of about 97,000 . The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively . These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.

Virus Res, 1988 Jan, 9(1), 11 - 20
Polyoma integrates readily in mouse cellular DNA; Roy G et al.; Although the natural host of polyoma virus is the mouse, its integration in cellular DNA has been investigated almost exclusively in rat cells . We report here studies on the integration of polyoma in mouse cells . We introduced the polyoma virus genome in two different mouse cell lines as an unselected genetic marker, by cotransfection with the tk gene of herpes simplex virus or the neo gene of E . coli . The number of TK+ or G418R clones obtained was reduced up to 50 fold by the presence of the polyoma genome . The gene coding for the early protein large T of polyoma was necessary and sufficient to produce this reduction . However, this effect appeared to be independent of polyoma replication . Surprisingly, all of the 33 clones analysed that had survived cotransfection with polyoma contained polyoma DNA integrated in their genome . Furthermore, in over 50% of these clones, the entire polyoma genome had been integrated . We conclude that polyoma integrates readily in mouse cellular DNA.

Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 334 - 8
Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector; Rekosh D et al.; A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector . The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication . Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gp120 in 20-30% of the transfected cells . By several criteria, the proteins were indistinguishable from those produced during infection . The proteins were localized to the cytoplasm and plasma membrane, and some of the gp120 was shed into the culture medium . Approximately 0.5 microgram of envelope protein could be extracted from 10(6) cells . This is at least 100 times higher than the levels found in HIV-infected H9 cells . In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed . Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.

Proc Natl Acad Sci U S A, 1988 Jan, 85(1), 83 - 7
Reversible receptor methylation is essential for normal chemotaxis of Escherichia coli in gradients of aspartic acid; Weis RM et al.; The chemotaxis of wild-type cells of Escherichia coli and double mutants lacking the methyltransferase and the methylesterase activities of the receptor modification system has been compared in spatial gradients of aspartic acid . Previous studies showing that a chemotactic response can be observed for the mutant raised questions about the role of methylation in the bacterial memory . To clarify the role of methylation, the redistribution of bacteria in stabilized defined gradients of aspartic acid was monitored by light scattering . There was no redistribution of the mutant cells in nonsaturating gradients of aspartic acid, but over the same range these mutant bacteria were observed to respond and to adapt during tethering experiments . In large saturating gradients of aspartate, slight movement of the mutant up the gradient was observed . These results show that dynamic receptor methylation is required for the chemotactic response to gentle gradients of aspartic acid and that methylation resets to zero and is part of the normal wild-type memory . There are certain gradients, however, in which the methylation-deficient mutants show chemotactic ability, thus explaining the apparent anomaly.

Mutat Res, 1988 Jan, 193(1), 75 - 86
The expression of the Escherichia coli uvrA gene in human cells; Dickstein R et al.; Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step . In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products . Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E . coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously . The E . coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells . The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E . coli gene is flanked by individual SV40 regulatory elements . SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt+ colonies were selected, and cell lines established . Several lines were examined in detail . Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95,000-dalton UvrA-related protein . The expression of uvrA was found to be 50-100-fold lower than the expression of gpt . Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities . The peptide maps derived from partial proteolysis of the "mammalian" UvrA protein are identical to the E . coli UvrA protein . The sub-cellular location of UvrA protein in uvrA+ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear . Two uvrA+ cell lines were examined for their UV-resistant phenotype and not unexpectedly were found not to be reverted to a state of repair proficiency.

J Bacteriol, 1988 Jan, 170(1), 89 - 97
Nucleotide sequence and expression of the aceK gene coding for isocitrate dehydrogenase kinase/phosphatase in Escherichia coli; Cortay JC et al.; The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylation-dephosphorylation of isocitrate dehydrogenase mediated by a bifunctional enzyme: isocitrate dehydrogenase kinase/phosphatase . The aceK gene coding for this enzyme is part of the polycistronic ace operon, which also includes the aceB and aceA genes coding, respectively, for malate synthase and isocitrate lyase, the two glyoxylate bypass enzymes . The complete nucleotide sequence of a 2,214-base-pair DNA fragment containing the aceK gene and its 5' flanking region has been determined . In vivo experiments based on gene expression in a minicell system and protein fusion with beta-galactosidase, as well as in vitro assays with a plasmid-directed transcription-translation coupled system, have shown that the aceK gene extends over 1,731 nucleotides encoding a 66,528-dalton protein . The 5' flanking region presents an unusual intercistronic structural pattern consisting of two consecutive long dyad symmetries, almost identical in sequence, which can yield very stable stem-loop units . These structures are probably responsible for the drastic downshifting in expression observed in acetate-grown bacteria between the aceK gene and the aceA gene located immediately upstream in the ace operon.

J Bacteriol, 1988 Jan, 170(1), 439 - 41
EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12; Slauch JM et al.; The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12 . Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ . In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein . These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.

J Bacteriol, 1988 Jan, 170(1), 386 - 92
Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map; Chung T et al.; In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass . The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK . We cloned this operon and determined its functional map by deletion analysis . The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques . The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined . Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis . In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable . This observation is consistent with a striking downshift in expression between aceA and aceK.

J Bacteriol, 1988 Jan, 170(1), 218 - 22
Activation of a cryptic gene by excision of a DNA fragment; Parker LL et al.; The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene . The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation . Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation . Our model for the retention of cryptic genes (B . G . Hall, S . Yokoyama, and D . H . Calhoun, Mol . Biol . Evol . 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele . We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+ . This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon . We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8) . We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.

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