Escherichia coli

J Biol Chem, 1994 Oct 7, 269(40), 24736 - 41
The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni; Tielens AG et al.; Hexokinase has been purified from adult Schistosoma mansoni worms and the activity shown to be associated with a single protein species having an M(r) about 50,000 . This protein is recognized on Western blots probed with antisera against rat Type I hexokinase or against a recombinant S . mansoni hexokinase that had been expressed in Escherichia coli using a previously cloned cDNA . An 18-residue N-terminal sequence determined for the purified S . mansoni hexokinase is identical to that deduced from the nucleotide sequence of the cDNA, consistent with the view that the cloned cDNA encodes the hexokinase characterized in the present study . The S . mansoni enzyme has a relatively low Km (approximately 60 microM) for glucose and is sensitive to inhibition (competitive versus ATP, Ki approximately 50 microM) by its product, glucose 6-phosphate (Glc-6-P) . With these kinetic properties and 50 kDa molecular mass, S . mansoni hexokinase resembles the ancestral hexokinase predicted to have given rise, by gene duplication and fusion, to the present day 100-kDa Glc-6-P-sensitive mammalian hexokinases . The schistosomal hexokinase represents the first 50-kDa Glc-6-P-sensitive hexokinase whose sequence has been obtained . The schistosomal hexokinase does not bind to mitochondria, consistent with its lack of a hydrophobic segment at the N terminus which is required for binding of the mammalian Type I and II isoenzymes to mitochondria . The marked Crabtree effect exhibited by S . mansoni cercariae may be at least partly attributed to the expression of rather high levels of a hexokinase having a high affinity for glucose but only a moderate sensitivity to product inhibition by Glc-6-P.

J Biol Chem, 1994 Oct 7, 269(40), 24608 - 14
Glutamic acid 86 is important for positioning the 80's loop and arginine 54 at the active site of Escherichia coli aspartate transcarbamoylase and for the structural stabilization of the C1-C2 interface; Baker DP et al.; Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's loop, which moves into the active site during the T to R transition . In order to determine whether this interaction is important for the correct positioning of the 80's loop and Arg-54 at the active site and also for the structural stabilization of the enzyme, a mutant version was created in which Glu-86 was replaced by Gln (Glu-86-->Gln) . Although the mutant holoenzyme exhibits almost normal homotropic cooperativity, both the holoenzyme and catalytic subunit exhibit substantial reductions in activity and affinity for aspartate and carbamyl phosphate . Furthermore, the mutant holoenzyme shows a marked decrease in the activation by ATP and by the bisubstrate analog N-(phosphonoacetyl)-L-aspartate, reduced inhibition by CTP, as well as reduced affinities for these ligands . Results from molecular dynamics simulations of the Glu-86-->Gln and Glu-86-->Ala enzymes suggest that the positions of the 80's loop and Arg-54 are significantly perturbed by the introduction of these mutations . Taken together, these results indicate that the interaction between Glu-86 and Arg-54 is important for the formation of the high affinity, high activity form of the enzyme by stabilizing the correct position of the 80's loop and Arg-54 at the active site . Heat inactivation experiments also demonstrated that Glu-86 plays a significant role in the structural stabilization of the C1-C2 interface, since the temperature required for loss of half of the activity of the Glu-86-->Gln catalytic subunit is reduced by 5 degrees C relative to the wild-type catalytic subunit.

J Biol Chem, 1994 Oct 7, 269(40), 24596 - 601
Carboxyl-terminal truncation of recombinant factor XIII A-chains . Characterization of minimum structural requirement for transglutaminase activity; Lai TS et al.; A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity . delta K513 and delta A502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the Km for the glutamine substrate and a 2-fold increase in the Km of the primary amine substrate . There was no detectable transglutaminase activity for either thrombin-activated delta Y481 or delta K462 . The rate of ammonia release of thrombin-activated delta K513 and delta A502 was reduced 6- and 4-fold, respectively, whereas ammonia release was not detected for the delta Y481 and delta 462 mutants . The Kact for calcium ions of the delta K513 mutant was similar to recombinant FXIIIa, whereas, it was increased by approximately 3-fold for the delta A502 mutant . The rate of fibrin gamma-chain dimer formation for the delta K513 and delta A502 mutants was reduced by approximately 19-fold . delta K462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound . In conclusion, these results documented that the carboxyl-terminal calcium binding domain (Asp468-Glu495) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred.

J Biol Chem, 1994 Oct 7, 269(40), 24586 - 95
Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase; Brennan TV et al.; The non-enzymatic deamidation at residues Asn-12 and Asn-38 of Escherichia coli phosphocarrier protein, HPr, and the repair of the resulting L-isoaspartyl (or beta-aspartyl) derivatives, HPr-1 and HPr-2, by recombinant human S-adenosylmethionine-dependent L-isoaspartate-(D-aspartate) O-methyltransferase (EC 2.1.1.77) were investigated . HPr is a component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is involved in the concomitant translocation and phosphorylation of many hexose sugars . The major products of the deamidation reaction, L-isoaspartyl (or beta-aspartyl) residues at positions 12 and 38, were found to be substrates for the L-isoaspartate-(D-aspartate) O-methyltransferase, an enzyme active on a wide variety of peptides and proteins containing these abnormal residues . This enzyme has been shown to catalyze the first step in a process that can convert L-isoaspartyl residues in peptides to normal L-aspartyl residues . The affinity of a recombinant human methyltransferase for HPr-1, a form deamidated at Asn-38, was relatively poor (Km = 3.6 mM), while a greater affinity was found for HPr-2, a form deamidated at both Asn-12 and Asn-38 (Km = 197 microM) . When HPr-2 was incubated with S-adenosylmethionine and the methyltransferase, the bulk of the L-isoaspartyl residues at position 12 was converted to L-aspartyl residues . The major-by-product was the D-isoaspartyl form . The conversion of L-isoaspartyl residues at position 38 to L-aspartyl residues was less complete, reflecting the lower affinity of the methyltransferase for this site . The phosphohydrolysis activity of the repaired form was found to be midway between the form containing only L-aspartyl residues at positions 12 and 38 and the deamidated HPr-2 form.

Nature, 1994 Oct 6, 371(6497), 528 - 31
Regulation of retinoid signalling by receptor polarity and allosteric control of ligand binding; Kurokawa R et al.; Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) regulate transcription by binding to response elements in target genes that generally consist of two direct repeat half-sites of consensus sequence AGGTCA (ref . 1) . RAR/RXR heterodimers activate transcription in response to all-trans or 9-cis retinoic acid by binding to direct repeats spaced by five base pairs (DR5 elements), such that RAR occupies the downstream half-site . RXR homodimers activate transcription in response to 9-cis retinoic acid by binding to direct repeats spaced by one base pair (DR1 elements) . Although RXR/RAR heterodimers bind to DR1 elements with higher affinity than RXR homodimers, in most contexts they are unable to activate transcription in response to either all-trans or 9-cis retinoic acid . As a result, RARs inhibit RXR-dependent transcription from these sites . We report that the switching of the RAR from an activator to an inhibitor of retinoid-dependent transcription requires that it be bound to the upstream half-site of DR1 elements and that it allosterically block the binding of ligand to the RXR.

Biochim Biophys Acta, 1994 Oct 6, 1214(3), 323 - 32
The effects of phosphoglycerides on Escherichia coli cardiolipin synthase; Ragolia L et al.; Escherichia coli cardiolipin synthase catalyzes the conversion of two phosphatidylglycerol molecules to cardiolipin and glycerol . This enzyme was amplified in strain BL21(DE3) bearing recombinant plasmid pLR3, which was itself constructed by inserting the cls gene downstream from a T7 RNA promoter . Membranes from BL21(DE3)/pLR3 have over 1200 times more cardiolipin synthase activity than do comparable membranes from wild type cells . The enzyme was purified to homogeneity by extraction with Triton X-114 and chromatography on DEAE-cellulose . The purified enzyme migrated as a single band (46 kDa) on SDS-PAGE . This, along with SDS-PAGE analysis of induced protein, supports the notion that cls is the structural gene for cardiolipin synthase . Cardiolipin synthase activity was determined in a mixed micelle assay in which phosphatidyl{2-3H}glycerol was the substrate . The enzyme is inhibited by the product of the reaction, cardiolipin, and by phosphatidate . However, it is not inhibited by two other anionic phosphoglycerides, phosphatidylinositol and bis-phosphatidate . Phosphatidylethanolamine partially offsets inhibition by cardiolipin but not by phosphatidate . Magnesium chloride has the opposite effect . Cardiolipin inhibition of cardiolipin synthase probably plays an important role in regulating cardiolipin synthesis in E . coli.

Biochemistry, 1994 Oct 4, 33(39), 11987 - 92
Mechanistic aspects of tagetitoxin inhibition of RNA polymerase from Escherichia coli; Mathews DE et al.; Tagetitoxin inhibits RNA synthesis directed by bacterial RNA polymerase, and the current study explores several mechanistic aspects of this inhibition . Tagetitoxin inhibition of in vitro RNA synthesis directed by Escherichia coli RNA polymerase is independent of the template DNA concentration . The toxin can block Escherichia coli RNA polymerase during elongation of a nascent RNA chain . In abortive initiation assays, the rate of dinucleotide formation is inhibited by tagetitoxin when initiated with ATP or CpA but not when AMP is used to initiate . Formation of longer oligonucleotides is inhibited by the toxin regardless of the initiating nucleotide . These abortive initiation studies indicate that tagetitoxin does not affect nucleotide substrate binding or phosphodiester bond formation and suggest that the toxin may interfere with a subsequent step . It is suggested that tagetitoxin affects the stability of nascent oligonucleotide binding and/or the translocation of the catalytic center with respect to the 3'-OH of nascent oligonucleotides.

Biochemistry, 1994 Oct 4, 33(39), 11971 - 9
Melting of a DNA helix terminus within the active site of a DNA polymerase; Hochstrasser RA et al.; Accurate synthesis of DNA by polymerase is due in part to the selective removal of misincorporated nucleotides by a 3'-5' exonuclease activity (proofreading) . Proofreading by an exonuclease domain containing a single-stranded DNA binding site may involve local melting of a duplex DNA substrate . Here we use time-resolved fluorescence spectroscopy to analyze the local melting of a DNA duplex terminus induced by the Klenow fragment of DNA polymerase I . Four oligodeoxynucleotide primer/templates were prepared, each containing the fluorescent adenine analog 2-aminopurine (A*) at the primer 3' terminus, and one of the common DNA bases opposite the A* residue . Fluorescence decays of the duplex DNAs and the single primer oligonucleotide were jointly analyzed using global analysis procedures . Four lifetime components were resolved in the duplex DNAs, representing distinct conformational states of the terminal A* residue: paired A* bases, partially stacked A* bases, and extended A* bases . The variation of the apparent fraction of paired A* bases with temperature was in accord with optical melting data, and the extent of base pairing observed in each duplex was consistent with the base-pairing preferences of A* established in other studies . These results establish that the fluorescence decay characteristics of A* can be used to examine base-pairing interactions at a DNA duplex terminus . Since the fluorescence of A* can be observed without interference from protein amino acid residues, unlike existing methods for monitoring DNA melting transitions, this method was used to examine the extent to which Klenow fragment could induce fraying at each duplex terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Oct 4, 33(39), 11927 - 34
Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms; Firestine SM et al.; A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E . coli and G . gallus has been conducted using in vivo and in vitro studies . In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) . The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-{(N-succinylamino)carbonyl}-5-aminoimidazole ribonucleotide (SAICAR) synthetase . The E . coli PurE and the C-terminal domain of the G . gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities . The substrate requirements of AIR carboxylases from E . coli and G . gallus have been compared by a series of in vitro experiments . The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E . coli PurE (Mueller et al., 1994) but not for the G . gallus AIR carboxylase . In contrast, AIR and CO2 are substrates for the G . gallus AIR carboxylase . The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR) . NAIR is a tight-binding inhibitor of the G . gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E . coli PurE . These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Oct 4, 33(39), 11917 - 26
Carboxylases in de novo purine biosynthesis . Characterization of the Gallus gallus bifunctional enzyme; Firestine SM et al.; Two successive steps in de novo purine biosynthesis are catalyzed by the enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-{(N-succinylamino)carbonyl}-5-aminoimidazole ribonucleotide (SAICAR) synthetase . Amino acid sequence alignments of the proteins from various sources suggested that several unusual differences exist within the structure and function of these enzymes . In vertebrates, a bifunctional enzyme (PurCE) catalyzes successive carboxylation and aspartylation steps of AIR to form SAICAR . This is in contrast to the three proteins, PurK, PurE, and PurC, from Escherichia coli which have recently been shown to require 2 equiv of ATP for the AIR to SAICAR conversion in the presence of physiological HCO3- concentrations (Meyer et al., 1992) . A comparative study of these proteins has been initiated using a high-production, heterologous expression system for the Gallus gallus AIR carboxylase-SAICAR synthetase and yields purified enzyme following a two-step procedure . Selective assays have been developed for all the enzymatic activities of the bifunctional protein . The G . gallus AIR carboxylase has no ATP dependence and displays a Km for HCO3- that is 10-fold lower than that for the related PurE protein from E . coli, supporting the hypothesis that the two enzymes require different substrates . No common cofactors or metals are required for catalysis . Each catalytic activity has been shown to be independent by selective inactivation of SAICAR synthetase with the affinity agent 5'-{4-(fluorosulfonyl)benzoyl}-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-binding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR) . The native protein aggregates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domains, each containing a different catalytic site.

Biochemistry, 1994 Oct 4, 33(39), 11868 - 74
Properties of tyrosine 766-->serine mutant of Escherichia coli DNA polymerase I: template-specific effects; Desai SD et al.; In order to determine the role of Tyr 766 of Escherichia coli DNA polymerase I in the catalysis of DNA synthesis, we investigated the properties of a Tyr 766-->Ser (Y766S) mutant of the Klenow fragment of E . coli DNA polymerase I . We found that the rates of incorporation of only dTTP but not the other dNTP substrates were affected in the reactions catalyzed by the mutant enzyme, when homopolymeric template-primers were used . The mutant enzyme exhibited a reduced rate of synthesis only with poly(rA)- or poly(dA)-directed reactions . Examination of the ability of the mutant and the wild-type enzymes to bind to dGTP and dTTP, as judged by UV-mediated cross-linking, indicated nearly identical binding efficiencies of both nucleotides . However, the ability of the mutant enzyme to bind to poly(rA).(dT)15 and poly(dA).(dT)15 was found to be significantly reduced as compared to the binding to heteropolymeric DNA . In order to further define the nature of template-mediated restriction on the catalytic activity of the mutant enzyme, its ability to copy DNA templates containing a stretch of AAAAA and ACACA sequences was compared . The results show that DNA synthesis catalyzed by the mutant enzyme is significantly retarded when it encounters the AAAAA region of the template but not the ACACA region . Product analysis of the reaction directed by the two template-primers showed that the mutant enzyme stalls/terminates synthesis upon encountering an AAAAA sequence in the template.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Oct 4, 33(39), 11767 - 75
Stability of myoglobin: a model for the folding of heme proteins; Hargrove MS et al.; Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells . The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission . Two key conclusions were reached . First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa . In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression . Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin . Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant . In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin . The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

Biochemistry, 1994 Oct 4, 33(39), 11692 - 8
Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase; Lindbladh C et al.; We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA- . The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl . The fusion protein produced was isolated and purified . Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000 . Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000) . This is the expected M(r) for the fusion protein subunit . The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes . In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined . It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed . In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.

FEBS Lett, 1994 Oct 3, 352(3), 318 - 22
Purification and spectroscopic characterization of a recombinant amino-terminal polypeptide fragment of mouse epithelial cadherin; Tong KI et al.; Cadherins are a family of Ca(2+)-dependent cell adhesion molecules containing four extracellular tandem repeats each of 110 amino acids . The most amino-terminal repeat is believed to confer the specificity of cell adhesion . A polypeptide containing the amino-terminal repeat of mouse epithelial cadherin has been over-expressed in E . coli and purified to homogeneity . This polypeptide binds Ca2+ with a dissociation constant of 1.6 x 10(-4) M . CD and NMR experiments indicate that the polypeptide adopts a predominantly beta-sheet conformation and that binding of Ca2+ induces only small conformational changes.

EMBO J, 1994 Oct 3, 13(19), 4695 - 703
Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli; Herrick J et al.; The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated . We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated . Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro . We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH) . The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane . A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication.

EMBO J, 1994 Oct 3, 13(19), 4670 - 5
A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis; Klebba PE et al.; LamB facilitates the uptake of maltose and maltodextrins across the bacterial outer membrane and acts as a general porin for small molecules . Using directed deletion mutagenesis we removed several regions of the LamB polypeptide and identified a polypeptide loop that both constricts the maltoporin channel and binds maltodextrins . In conjunction with a second sugar binding site that we identified at the rim of the channel, these data clarify, for the first time, the mechanism of transport through a substrate-specific porin . Furthermore, unlike the transverse loops of general porins, which originate from a central location in their primary structure, the loop that regulates LamB permeability originates from a C-terminal site . Thus LamB represents a second distinct class of porins in the bacterial outer membrane that is differently organized and separately evolved from OmpF-type, general porins.

EMBO J, 1994 Oct 3, 13(19), 4662 - 9
Translocation of N-terminal tails across the plasma membrane; Cao G et al.; Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N-terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli . We have now examined the mechanism of insertion of N-terminal periplasmic tails and have defined the features needed to translocate these regions . We find that short tails of up to 38 residues are efficiently translocated in a SecA- and SecY-independent manner while longer tails are very poorly inserted . Efficient translocation of a 138 residue tail is restored and is Sec-dependent by the addition of a leader sequence to the N-terminus of the protein . We also find that while there is no amphiphilic helix requirement for N-terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region . Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail.

EMBO J, 1994 Oct 3, 13(19), 4558 - 67
CRP induces the repositioning of MalT at the Escherichia coli malKp promoter primarily through DNA bending; Richet E et al.; Activation of transcription initiation at the Escherichia coli malKp promoter requires the repositioning of MalT, the primary activator, from a set of non-productive sites to a set of productive sites, which is staggered by 3 bp . Occupation of the latter relies on the formation of a higher order structure involving distal MalT sites and the binding of CRP (cAMP receptor protein) to three sites located in the intervening region . We show here that one can successfully replace all of the CRP sites by the binding site of another DNA-bending protein, integration host factor, or by a sequence-directed bend without altering the process of malKp activation . This observation indicates that CRP action at malKp does not involve critical interactions with MalT and that CRP promotes MalT repositioning primarily through DNA bending . This structural role of CRP differs markedly from its role in the activation of the lac promoter.

EMBO J, 1994 Oct 3, 13(19), 4549 - 57
The functional subunit of a dimeric transcription activator protein depends on promoter architecture; Zhou Y et al.; In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase . In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site . We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase {CC(-61.5) and CC(-72.5)} and at one Class II promoter {CC(-41.5)} . Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP . In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP.

EMBO J, 1994 Oct 3, 13(19), 4536 - 48
Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins; Segall AM et al.; Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination . HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity . We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase . The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA . We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure . We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome . HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions . These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts . Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA . While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies . This probably reflects subtle structural differences between the assembled complexes.

Virology, 1994 Oct, 204(1), 420 - 4
The protein p30, encoded at the gag-pro junction of mouse mammary tumor virus, is a dUTPase fused with a nucleocapsid protein; Bergman AC et al.; A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30 . The protein consists of two domains, the zinc-finger-containing nucleocapsid (NC) protein portion with 95 residues and a C-terminal extension comprising 154 residues . The C-terminal domain shows similarity in sequence with the enzyme dUTPase from other sources . In this paper, we demonstrate that p30 is a functional dUTPase . Overproduction of the NC protein in Escherichia coli, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of readthrough . By a 1-base insertion, eliminating the gag stop codon and fusing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed . The overproduced protein, purified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity . Analytical gel filtration and sequence homology to other dUTPases suggest a trimeric assembly of p30 subunits . MMTV thus possesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain of dUTPase.

Virology, 1994 Oct, 204(1), 376 - 90
Vaccinia virus gene A36R encodes a M(r) 43-50 K protein on the surface of extracellular enveloped virus; Parkinson JE et al.; A characterization of vaccinia virus strain Western Reserve (WR) open reading frame (ORF) A36R is described . This ORF is predicted to encode a 221-amino-acid protein (M(r) 25.1 K) with an amino-terminal hydrophobic sequence, seven potential sites for attachment of N-linked carbohydrate, but no carboxy-terminal transmembrane anchor . It is identical in vaccinia strain Copenhagen and shares 94.6% amino acid identity with the corresponding ORF in variola virus strains Harvey, India-1967, and Bangladesh-1975 . RNA analyses detected a 600-nucleotide, early transcript that initiated 10-13 nucleotides upstream of the A36R ORF, and heterogeneously sized late transcripts running across the ORF . A rabbit antiserum raised against an Escherichia coli glutathione S-transferase fusion protein identified M(r) 43-50 K proteins that accumulated late during vaccinia virus infection and fractionated as integral membrane proteins during Triton X-114 partitioning . Similar polypeptides were expressed by vaccinia virus strains Tian Tan, Tashkent, Lister, Wyeth, Copenhagen, and IHD-J and by rabbitpox virus and cowpox virus (strain Brighton Red) . Immunoblot analysis of purified and protease-digested intracellular mature virus (IMV) and extracellular enveloped virus (EEV) showed that the A36R proteins were present on the surface of EEV with type II membrane topology, but were absent from IMV . A WR deletion mutant lacking the A36R ORF (delta A36R) had a small plaque phenotype on all cell lines tested . IMV formation by delta A36R was unaltered but EEV formation was reduced approximately fivefold compared to wild-type (WT) when measured either by density gradient analysis of isotopically labeled virions or by infectivity assays . Thus the loss of the A36R protein from the EEV surface did not reduce EEV specific infectivity in vitro . Despite this, delta A36R showed striking attenuation compared with WT in a murine intranasal model . Finally, a revertant virus in which the A36R ORF was restored showed WT plaque size, EEV formation, and virulence, demonstrating that all the phenotypic differences of delta A36R were attributable to loss of the A36R gene and not to other mutations acquired during its construction.

Virology, 1994 Oct, 204(1), 242 - 50
Identification and characterization of Marek's disease virus genes homologous to ICP27 and glycoprotein K of herpes simplex virus-1; Ren D et al.; We have identified two Marek's Disease Virus (MDV) genes within the EcoRI-B fragment of MDV-GA genomic DNA . EcoRI-B is 11.3-kb long and maps within the long unique region of MDV genomes . A 3.2-kb fragment of EcoRI-B has been sequenced and contains two open reading frames, ORF53 and ORF54 . ORF53 (MDV gK), a homolog to HSV-1 glycoprotein K (gK), is 1062 nucleotides long and encodes 354 amino acids (39.5 kDa) . ORF54, designated MDV ICP27, based on significant similarity to HSV-1 ICP27, is 1419 nucleotides long and encodes 473 amino acids (54.5 kDa) . In Northern blot hybridization, two overlapping transcripts (2.9 and 1.6 kb) were detected in MDV-infected DEF cells treated with cycloheximide, suggesting that both transcripts belong to the immediate-early gene family . Amino acid sequence analysis of MDV gK shows some common glycoprotein features, including a putative N-terminal signal sequence, four N-linked glycosylation sites, and four potential transmembrane domains . Comparison of the predicted amino acid sequence of MDV ICP27 with that of HSV-1 ICP27 and VZV ORF4 shows a high degree of conservation within the C-terminus . The C-terminal region of HSV-1 ICP27 has been demonstrated to be critical to its function . A conserved zinc finger metal-binding motif C(442)-X4-C(447)-X13-H(461)-C(467) was also found in the C-terminus of MDV ICP27 . Furthermore, MDV ICP27 upstream sequences contain four copies of consensus sequence elements similar to the tegument protein target sequence TAATGARAT . TrpE-ICP27 fusion protein was expressed in Escherichia coli, and rabbit antisera were generated using purified fusion protein . A 55-kDa protein has been detected in both MDV-GA- and Md11-infected cells using immunoblot analysis.

J Virol, 1994 Oct, 68(10), 6487 - 95
Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus; Boniotti B et al.; Expression studies conducted in vitro and in Escherichia coli led to the identification of a protease from rabbit hemorrhagic disease virus (RHDV) . The gene coding for this protease was found to be located in the central part of the genome preceding the putative RNA polymerase gene . It was demonstrated that the protease specifically cuts RHDV polyprotein substrates both in cis and in trans . Site-directed mutagenesis experiments revealed that the RHDV protease closely resembles the 3C proteases of picornaviruses with respect to the amino acids directly involved in the catalytic activity as well as to the role played by histidine as part of the substrate binding pocket.

J Virol, 1994 Oct, 68(10), 6411 - 20
The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID; Tan SH et al.; The E6 promoters of all genital human papillomaviruses have a characteristic alignment of transcription factor binding sites . Activation of the basic transcription complex at the TATA box depends upon a sequence-aberrant Sp1 site . Repression of E6 promoters is achieved by two binding sites for the viral E2 protein positioned between the Sp1 site and the TATA box . We have purified the human papillomavirus type 16 E2 protein after expression in Escherichia coli and studied its binding and repression properties with oligonucleotides representing the homologous promoter sequences . A Kd value of 3 x 10(-10) M indicated binding properties expected for a native protein . We found low cooperativity in the binding of two E2 dimers to flanking sites, both when these sites were separated by 3 nucleotides, as in the natural promoter, and when they were further apart . E2 protein, bound close to the distal Sp1 site, displaced the Sp1 factor even when the aberrant sequence was replaced by a typical Sp1 core recognition site . The high affinity of E2 protein for its binding site even led to Sp1 displacement at concentrations of E2 protein nearly 2 orders of magnitude lower than those of Sp1 . Functional analyses of mutated E6 promoter sequences showed repression by this distal E2 binding site in the complete absence of binding to the proximal E2 binding site . From our findings and observations published by others, we conclude that each of the E2 binding sites in the E6 promoter of genital human papillomaviruses plays a separate role by displacing the transcription factors Sp1 and TFIID.

J Virol, 1994 Oct, 68(10), 6243 - 53
Peripheral blood mononuclear phagocytes mediate dissemination of murine cytomegalovirus; Stoddart CA et al.; Cytomegalovirus is transmitted with blood and organs from seropositive individuals, although the particular leukocyte population harboring latent or persistent virus remains poorly characterized . Murine cytomegalovirus, tagged with the Escherichia coli lacZ gene, was used to identify cells in which virus replicates during acute infection of immunocompetent mice . Recombinant murine cytomegaloviruses, RM461, RM460, and RM427, were constructed to express beta-galactosidase under control of the human cytomegalovirus ie1/ie2 promoter/enhancer . The lacZ gene was inserted between the ie2 and sgg1 genes in RM461 and RM460, disrupting a 0.85-kb late transcript that was found to be dispensable for replication in cultured cells as well as for infection of mice . In BALB/c mice, lacZ-tagged and wild-type viruses exhibited a similar 50% lethal dose and all had the capacity to latently infect the spleen . Peripheral blood mononuclear phagocytes were the major infected leukocyte cell type, as demonstrated by the ability of infected cells to adhere to glass and to phagocytize latex beads; however, these cells did not exhibit typical monocyte markers . Plaque assay for virus and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of frozen sections of organs from infected mice revealed that the major target organs included the spleen, adrenal glands, liver, and salivary glands, although tissues as diverse as brown fat and lungs were also involved . Individual blue-staining cells were readily identified in all infected tissues . These studies identified a mononuclear phagocyte, possibly a macrophage or dendritic cell precursor, as the vehicle of virus dissemination during acute infection, and demonstrate the utility of using lacZ-tagged murine cytomegalovirus for tropism, pathogenesis, and latency studies.

Microbiology, 1994 Oct, 140 ( Pt 10), 2543 - 53
Isolation of the Thiobacillus ferrooxidans ntrBC genes using a T . ferrooxidans nifH-lacZ fusion; Kilkenny CA et al.; An agar plating technique was developed in which the activation of expression of a Thiobacillus ferrooxidans nifH-lacZ gene fusion was used to isolate the ntrBC genes from a T . ferrooxidans gene library . An Escherichia coli ntrC mutant containing the nifH-lacZ fusion was transformed and plated on a low-nitrogen medium so that on flooding with ONPG, the production of yellow colonies indicated the presence of the cloned T . ferrooxidans ntrBC genes . A 4.47 kb region from the T . ferrooxidans chromosome was sequenced . Analysis of the sequence revealed that the ntrB and ntrC genes were closely linked to a third ORF of unknown function . Analysis of the 900 bp region upstream of the T . ferrooxidans ntrBC genes and Southern hybridization experiments confirmed that in T . ferrooxidans ATCC 33020, the glnA and ntrBC genes are unlinked . Expression of the T . ferrooxidans nifH-lacZ fusion in E . coli was activated in the presence of the T . ferrooxidans ntrBC genes and regulated by nitrogen.

Microbiology, 1994 Oct, 140 ( Pt 10), 2531 - 41
Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli; Gruer MJ et al.; An acnA mutant of Escherichia coli was constructed by replacing the chromosomal acnA gene by an internally deleted derivative containing a kanR cassette . Southern and Western blotting confirmed that the acnA gene had been replaced by the disrupted gene and that the aconitase A protein was no longer expressed . However, the mutant failed to exhibit the anticipated glutamate auxotrophy and it retained a residual aconitase activity . This activity was due to an analogous unstable enzyme(s) designated aconitase B . Studies on the regulation of aconitase A synthesis using an acnA-lacZ translational fusion showed that the acnA gene resembles other citric acid cycle genes in being subject to CRP-mediated catabolite repression and ArcA-mediated anaerobic repression . In addition to being activated by the SoxRS oxidative stress regulatory system, the acnA gene appeared to be activated by the ferric uptake regulator (Fur) . It was concluded that the acnA gene belongs to at least four global regulatory networks, crp, arcA, fur and soxRS . In contrast, the aconitase B activity decreased after exposure to oxidative stress and was less affected by anaerobiosis . Comparable studies with the fumarase genes (fumA, B and C) indicated that fumA (encoding the unstable aerobic iron-sulphur-containing fumarase) is activated by the ferric uptake regulator (Fur) and fumC (encoding the stable fumarase) is activated by the SoxRS oxidative stress regulatory system.

J Gastroenterol, 1994 Oct, 29(5), 631 - 6
Small duct cholangitis induced by N-formyl L-methionine L-leucine L-tyrosine in rats; Yamada S et al.; Primary sclerosing cholangitis (PSC) frequently accompanies inflammatory bowel diseases . In an attempt to increase our understanding of the pathogenesis of PSC, we studied bile duct changes in rats with colitis which had been given N-formyl L-methionine L-leucine L-tyrosine (fMLT) rectally; fMLT is one of the chemotactic peptides produced by Escherichia coli, and is secreted into the bile by hepatocytes after it enters the portal blood . Transrectal administration of fMLT induced a marked inflammation in the portal triad and mild hepatocyte necrosis on the 4th day . The infiltrating leukocytes in the portal tract were mostly mononuclear cells, which densely infiltrated around the bile ducts . These mononuclear cells appeared to attach to bile duct epithelial cells, and they were more numerous in the smaller bile ducts . Electron microscopy revealed that lymphocytes were in direct contact with bile duct lining cells and that some epithelial cells had degenerated or collapsed . These results suggest that this E . coli-derived peptide may induce cholangitis in the small bile duct through cell-mediated mechanisms . Since these pathologic changes resemble those of the bile duct observed in the early stage of PSC, it can be concluded that bacterial chemotactic peptides may play a role in the pathogenesis of small-duct PSC.

Int J Exp Pathol, 1994 Oct, 75(5), 363 - 8
Interferon-gamma and polyunsaturated fatty acids increase the binding of lipopolysaccharide to macrophages; Darmani H et al.; We have previously shown that interferon-gamma (IFN-gamma) increases the polyunsaturated fatty acid content of membrane phospholipids in cells that were sensitive to endotoxin . In this study, IFN-gamma was found to stimulate the binding of endotoxin to the murine macrophage cell line J774.2 and the human monocyte cell line U937 . Interferon-gamma-activated J774.2 cells showed a 66% increase in fluoresceine isothiocyanate (FITC) labelled LPS binding (P < 0.0005 vs control cells) and a 49% increase in tritium labelled LPS binding (P < 0.0001 vs control cells) . Interferon-gamma also induced a 35% increase in binding of FITC-LPS in U937 cells (P < 0.0001 vs control cells) . In contrast, pretreatment of J774.2 cells with interferon-beta (IFN-beta) had no effect on binding of FITC-LPS . Preincubation with exogenously supplied polyunsaturated fatty acids, linoleic and arachidonic acids, resulted in increases of 74% and 69% in FITC-LPS binding, respectively (both P < 0.0005 vs control cells) . On the other hand, pretreatment with the saturated fatty acid, palmitic acid, had no effect on FITC-LPS binding . We propose that IFN-gamma-induced changes in the membrane phospholipid fatty acid composition of macrophage-like cells influence the binding of endotoxin.

Liver, 1994 Oct, 14(5), 230 - 3
Endotoxin-induced defenestration of the hepatic sinusoidal endothelium: a factor in the pathogenesis of cirrhosis?
Dobbs BR, Rogers GW, Xing HY, Fraser R.
Defenestration and capillarisation of the hepatic sinusoidal endothelium occurs early in the pathogenesis of cirrhosis, both in patients suffering from alcohol abuse and in animal models . It is possible also that alcohol abuse promotes the absorption of bacterial endotoxins from the gastrointestinal tract . In this study we have investigated the effects of a single intravenous injection of endotoxin on the hepatic sinusoidal endothelium of rats . Seven days after the dose of endotoxin, the porosity of the sinusoidal endothelium was reduced to 40% of that of controls, due to a decrease in both diameter and number of fenestrae . The livers examined 14 days after dosing exhibited normal porosity . We postulate that bacterial endotoxins play a role in the pathogenesis of cirrhosis by modulating the fenestrated sinusoidal endothelium (liver sieve).

Plant Cell, 1994 Oct, 6(10), 1495 - 507
The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast; Dewey RE et al.; Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine . A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1 . The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase) . Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline . Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library . Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system . The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.

Photochem Photobiol, 1994 Oct, 60(4), 295 - 300
Photosensitized formation of 8-hydroxy-2'-deoxyguanosine and DNA strand breakage by a cationic meso-substituted porphyrin; Nicotera TM et al.; Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA . In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic porphyrin . The data demonstrated a dose-dependent formation for each type of DNA damage . Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage . However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D2O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process . These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity.

FEMS Microbiol Lett, 1994 Oct 1, 122(3), 297 - 302
Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane; Hitotsubashi S et al.; The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine . The binding of 125I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific . Proteins cross-linked with 125I-STII were purified by column chromatography on hydroxyapatite and TSK gel . Analyses of the purified protein by SDS-polyacrylamide gel electrophoresis and gel filtration showed that the molecular mass was 25 kDa.

Appl Environ Microbiol, 1994 Oct, 60(10), 3862 - 3
Immobilization of Escherichia coli expressing the lux genes of Xenorhabdus luminescens; Marincs F et al.; The luxCDABE operon of Xenorhabdus luminescens was cloned into pUC18 to make pLITE27 . Expression of the lux genes from the lac promoter resulted in strong constitutive light emission by Escherichia coli DH5 carrying the recombinant lux plasmid, pLITE27 . When strain DH5(pLITE27) was immobilized with sodium alginate-CaCl2, the embedded cells retained their luminescence up to 2 weeks under appropriate storage conditions.

Appl Environ Microbiol, 1994 Oct, 60(10), 3724 - 31
Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110; Varma A et al.; Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions . These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design . Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models . In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110 . We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g {dry weight} per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g {dry weight} per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g {dry weight} per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g {dry weight} per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass) . The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments . We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures . In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium . In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1994 Oct, 60(10), 3503 - 7
Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon; Selifonova OV et al.; The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14 . Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615 . The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E . coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM . After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+ . The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions . The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+ . External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions . Sodium ions appear to facilitate mercury transport . We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E . coli and that the Na+ gradient may energize the transport of Hg2+.

Appl Environ Microbiol, 1994 Oct, 60(10), 3485 - 90
Production and release of polyphosphate by a genetically engineered strain of Escherichia coli; Hardoyo et al.; A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium . PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy . Once E . coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake . PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis . PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium . When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed . Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C . These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E . coli recombinants . High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E . coli recombinant . The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium . Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.

Bioessays, 1994 Oct, 16(10), 761 - 6
Endo-exonucleases: enzymes involved in DNA repair and cell death?
Fraser MJ.
Endo-exonucleases from E . coli to man, although very different proteins, are multifunctional enzymes with similar enzymatic activities . They probably have two common but opposing biological roles . On the one hand, they promote survival of the organism by acting in recombination and recombinational DNA repair to diversify and help preserve the genome intact . On the other hand, they degrade the genomic DNA when it is damaged beyond repair . This ensures elimination of heavily mutagenized cells from the population.

J Mol Evol, 1994 Oct, 39(4), 340 - 6
Compositional heterogeneity of the Escherichia coli genome: a role for VSP repair?
Gutierrez G, Casadesus J, Oliver JL, Marin A.
E . coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers) . Conversely, E . coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers) . These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies . Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity . The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample . This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample . A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI) . A possible link between the rate of VSP activity and the level of gene expression is considered.

Gut, 1994 Oct, 35(10), 1449 - 54
Adhesion of human enterotoxigenic Escherichia coli to human mucus secreting HT-29 cell subpopulations in culture; Kerneis S et al.; Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations . These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively . Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed . All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity . Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation . The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity . Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border . Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins.

Genes Dev, 1994 Oct 1, 8(19), 2363 - 74
Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization; Weinreich MD et al.; The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans . In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay . Two mutations in the carboxyl terminus were isolated repeatedly . The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans . The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans . Combining both mutations increases Tnp activity 80-fold . Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh . Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers . Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization . In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.

Dev Biol, 1994 Oct, 165(2), 639 - 53
Transcriptional and post-transcriptional regulation of maternal and zygotic cytoskeletal tropomyosin mRNA during Drosophila development correlates with specific morphogenic events; Hales KH et al.; We have characterized maternal and zygotic cytoskeletal tropomyosin mRNA expression during Drosophila embryogenesis using in situ hybridization to endogenous cytoskeletal tropomyosin mRNA and a cytoskeletal tropomyosin promoter/beta-galactosidase-encoding fusion gene mRNA in transgenic flies . A 2.0-kb maternal cytoskeletal tropomyosin mRNA is synthesized in the nurse cells and transported into the oocyte during oogenesis . During early embryogenesis, this mRNA becomes localized to the pole cell region and then to the cortex during the cellular blastoderm stage . In later embryos it is localized to the ventral and cephalic furrows and extending germ band . The major zygotic mRNA is 2.4 kb and is first detected at gastrulation . In early embryos, this mRNA is expressed in the invaginating anterior and posterior midgut, the transverse furrows, and the amnioserosa, all regions of the embryo undergoing intense cellular movement, and in later embryos, predominantly in the gut, brain, and epidermis . A transgene construct containing 1.2 kb of 5' cytoskeletal tropomyosin promoter sequences driving expression of Escherichia coli beta-galactosidase and the cytoskeletal tropomyosin 3' untranslated region in transgenic flies has the same distribution of maternal and zygotic transcripts throughout oogenesis and embryonic development as the endogenous transcripts . A transgene containing the 3' untranslated region from either the hsp70 gene or the SV40 early genes, on the other hand, does not express maternal RNA . Furthermore, none of the transgenes expressed in the follicle cells, suggesting that expression in these cells is under different transcriptional control . Our results indicate that maternal and zygotic cytoskeletal tropomyosin mRNAs are localized to specific regions of the developing embryo, particularly in regions of the embryo undergoing cell movement and invagination . Furthermore, the synthesis, transport, and accumulation of this RNA is under transcriptional and post-transcriptional control.

Carcinogenesis, 1994 Oct, 15(10), 2143 - 8
An assay for O6-alkylguanine-DNA alkyltransferase based on restriction endonuclease inhibition and magnetic bead separation of products; Wilson BD et al.; We describe an improved, rapid and cost effective assay for measuring O6-alkylguanine-DNA alkyltransferase (AGT) levels in large numbers of small biological samples . The assay is based on the use of a synthetic O6-methylguanine oligonucleotide bound to magnetic beads via a biotin-streptavidin linkage and bound to its complement . A 35S label is incorporated into the free end of the duplex . The basis of the assay lies in the observation that the restriction enzyme PvuII will not cleave the bead-bound duplex oligonucleotide having an O6-methylguanine within the restriction sequence . However, on removal of the methyl group by AGT present in cell extracts prior to incubation with PvuII, the 35S-labelled oligonucleotide is cleaved by the restriction enzyme, releasing a 35S-labelled 8 bp fragment . Due to the stoichiometric nature of the AGT reaction, quantitation of AGT is easily achieved by counting an aliquot of the supernatant after pelleting the unrestricted bead complexes with a magnet . AGT levels measured in certain cell lines and human lymphocytes by the reported assay are comparable to other methods . The assay can be performed in basic laboratories and allows for the rapid processing of many samples simultaneously, which could prove useful in clinical and epidemiological studies.

Carcinogenesis, 1994 Oct, 15(10), 2125 - 9
The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo; Wink DA et al.; Nitric oxide has been shown to be a mediator molecule in the regulation of many physiological functions . However, this small diatomic molecule in the presence of O2 generates reactive intermediates which modify DNA bases and inactive enzymes at high concentrations (100 microM) . We report that NO generated by 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO, Et2NN(O)NO-Na+), a compound known to release NO in a predictable manner, caused irreversible damage at physiological concentrations to the zinc finger-containing DNA repair enzyme formamidopyrimidine-DNA glycolyase (Fpg protein) . The inhibition of the enzyme activity was DEA/NO dose and time dependent with IC50s with respect to total NO released from this compound of approximately 110 and approximately 120 mumol/l respectively . This inhibitory effect by P3 was not reversible over time in the presence of reducing agents and/or Zn2+ . Nitrite and diethylamine, the nitrogenous products of the decomposition of DEA/NO, did not inhibit the enzyme . The presence of 500 micrograms/ml bovine serum albumin did not protect the protein from the inhibitory effects of DEA/NO, however, the presence of 10 mM cysteine did dramatically abate the inhibition of the Fpg protein by DEA/NO . Other DNA glycosylases tested were not inhibited by exposure to these concentrations of NO . These results, together with reports of site-directed mutagenesis of this protein, suggest that the cysteine residues contained within the zinc finger motif of the Fpg protein are the primary sites of NO interaction . Our studies were then extended to intact cells . The Fpg protein activity was decreased following treatment in vivo when Escherichia coli MH321 (acr A-) cells were treated with DEA/NO . Furthermore, the Fapy-DNA glycosylase activity in H4 cells, a rat hepatoma line, was decreased when intact cells were incubated with DEA/NO.

Plant Mol Biol, 1994 Oct, 26(2), 747 - 56
Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence; Keller B et al.; The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco . Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems . A 169 bp fragment (-205 to -36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (-205 to -64) strongly activated these minimal promoters both in vascular and cortical cells . These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between -64 and -36 . The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations . A 24 bp sequence (bp -119 to -96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (-98 to -73, corresponding to the RSE regulatory element) induced vascular-specific expression . Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression.

Plant Mol Biol, 1994 Oct, 26(2), 699 - 708
Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo; Wada M et al.; Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction . The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases . One transmembrane domain had a leucine zipper structure . HAA1 was not a full-length gene (2054 bp) and lacked the 5' region, but it also included the conserved regions and putative transmembrane domains . Antibodies against the regions and putative transmembrane domains . Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H . akashiwo cells . Immunostaining of H . akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles.

Plant Mol Biol, 1994 Oct, 26(2), 679 - 90
Identification of a maize root transcript expressed in the primary response to nitrate: characterization of a cDNA with homology to ferredoxin-NADP+ oxidoreductase; Ritchie SW et al.; To more fully understand the biochemical and molecular events which occur in plants exposed to nitrate, cDNAs whose accumulation was enhanced in nitrate- and cycloheximide-treated maize (Zea mays L . W64A x W182E) roots were isolated . The 340 bp Zmrprn1 (for Zea mays root primary response to nitrate) cDNA also hybridized with a probe enriched for nitrate-induced sequences, and was characterized further . Sequence analysis of a near full-length cDNA (Zmrprn1A) showed strong homology (> 90% amino acid identity) with a root ferredoxin-NADP+ oxidoreductase (FNR) of rice, and 45-50% amino acid identify with leaf FNR genes . When expressed in Escherichia coli, the Zmrprn1A cDNA produced a protein with NADPH: ferricyanide reductase activity, consistent with the enzymatic properties of an FNR . The Zmrprn1 cDNA hybridized with a 1.4 kb transcript which was expressed in the maize root primary response to nitrate . That is, mRNA levels in roots increased rapidly and transiently in response to external nitrate, and low levels of nitrate (10 microM) induced transcript accumulation . The accumulation of the Zmrprn1 transcript was not prevented by cycloheximide, indicating that the cellular factor(s) required for expression were constitutively present in maize roots . The Zmrprn1 mRNA accumulated specifically in response to nitrate, since neither K+ nor NH4+ treatment of roots caused transcript accumulation . Maize leaves had about 5% of the transcript level found in roots, indicating a strong preference for expression of Zmrprn1 in roots . Analysis of maize genomic DNA indicated the presence of only a single gene or very small gene family for the Zmrprn1 . Together, the data indicate that Zmrprn1A encodes a nitrate regulated maize root FNR.

Plant Mol Biol, 1994 Oct, 26(1), 423 - 34
Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression; Kim DJ et al.; A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth . One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway . The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively . The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein . The antibody recognises a single polypeptide of ca . 74 kDa, in extracts of cotyledons, leaves and roots . The cucumber genome contains a single pck gene . In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease . Both accumulate again to a low level in senescing cotyledons . This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS) . When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not . Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised . While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.

Plant Mol Biol, 1994 Oct, 26(1), 353 - 62
Arabidopsis heat shock factor: isolation and characterization of the gene and the recombinant protein; Hubel A et al.; We have isolated the Arabidopsis heat shock factor gene Athsf1 as genomic and corresponding cDNA sequences via cross-hybridization with tomato clones . Sequence analysis indicates only a partial homology with the HSFs from tomato and other organisms which is confined to the DNA-binding and the oligomerization domains . The gene is constitutively expressed but the level of mRNA for Athsf1 increases two-fold upon heat shock . However, the putative promoter region lacks the canonical heat shock elements . After expression in Escherichia coli the recombinant Athsf1 protein binds specifically to a synthetic oligonucleotide containing five heat shock elements . The native size of recombinant ATHSF1 in vitro is consistent with a trimer as demonstrated by chemical cross-linking and pore exclusion limit analysis.

Plant Mol Biol, 1994 Oct, 26(1), 225 - 34
Purification and characterization of pea thioredoxin f expressed in Escherichia coli; Hodges M et al.; The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide . This cDNA fragment was subcloned into a pET expression vector and used to transform E . coli cells . After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells . The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity . The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin . The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay . Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E . coli . At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.

Plant Mol Biol, 1994 Oct, 26(1), 211 - 23
Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases; Brown AP et al.; We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity . A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated . Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells . Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells . Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays . The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543 . The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids . Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E . coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.

Plant Mol Biol, 1994 Oct, 26(1), 117 - 30
Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein; Betts SD et al.; The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII) . Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana . Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration . In cells grown under weak aeration, the mature protein accumulated upon induction . In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased . Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor . Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein . We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217-227).

Biopolymers, 1994 Oct, 34(10), 1339 - 47
Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering; Moradian-Oldak J et al.; Recombinant murine amelogenins M179 and M166 were expressed in Escherichia coli and purified . The aggregation properties of these amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering . Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight . Polydispersity and statistical parameters of how to interpret the analysis are also provided . Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations . It was shown that at pH 7.8-8 and ionic strength of 0.02-0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm . Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of amelogenin molecules to form aggregates . Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179.

Arthritis Rheum, 1994 Oct, 37(10), 1445 - 52
Analysis of the specificity of anti-PM-Scl autoantibodies; Ge Q et al.; OBJECTIVE . To compare the specificity of anti-PM-Scl autoantibodies in serum samples from 43 patients with myositis, scleroderma, or both . METHODS . Anti-PM-Scl immunoprecipitates from HeLa cell extract were used as antigen for immunoblot analyses to determine the antigenic components . A series of complementary DNA fragments was expressed in Escherichia coli for immunoblot examination of the reaction with the 100-kd protein . RESULTS . The immunoblot against immunoprecipitates was sensitive and specific for detecting reactions with components of the PM-Scl antigen: 42 of 43 sera (97.7%) reacted with the 100-kd, 27 of 43 (62.8%) with the 70-kd, and 5 of 43 (11.6%) with the 37-kd protein (not previously recognized as antigenic) . Forty-one sera reacted with N-terminal protein S1 (amino acids 11-437), 39 with central protein S2 (amino acids 439-749), and 24 with C-terminal protein S3 (amino acids 750-882) . Of 42 sera tested, 28 (66.7%) reacted most strongly with S1, and 6 (14.3%) reacted most strongly with S2 . Absorption studies implied additional, conformational epitopes not present on the bacterially expressed antigen . CONCLUSION . There was an overall similarity in reactivity to the PM-Scl antigen, but there were differences in the reactivity to the 70-kd and 37-kd proteins, as well as in the relative strength of the reactivity to the S2 protein.

Biochem J, 1994 Oct 1, 303 ( Pt 1), 73 - 9
Expression of a biologically active plant cytochrome b5 in Escherichia coli; Smith MA et al.; Cytochrome b5 from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff (1990) (Methods Enzymol . 185, 60-89) . Transformed cells were red in colour and accumulated cytochrome b5 to a level of around 30% of the total cell protein . The purified cytochrome had oxidized, reduced and low-temperature absorbance spectra characteristic of plant microsomal cytochrome b5, and exhibited a c.d . spectrum resembling that of a mammalian cytochrome b5 . The recombinant protein appeared to be correctly assembled and biologically active, being reduced by NADH in the presence of microsomal membranes prepared from the developing seeds of sunflower (Helianthus annuus) . Inhibition of haem synthesis in the transformed E . coli cells expressing cytochrome b5, by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b5 holoprotein but had little effect on the accumulation of cytochrome apoprotein . The recombinant protein expressed in E . coli therefore has the biochemical features of the higher-plant cytochrome b5 and can be used in studies of plant microsomal oxidation/reduction reactions.

Arch Biochem Biophys, 1994 Oct, 314(1), 132 - 41
Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties; Haggett KD et al.; A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli . The method affords substantial amounts (approx . 80 mg) of homogeneous enzyme . We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates . Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates . Mutant 55 had broad specificity and high activity . In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease . The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues . Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower . Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue.

Am J Obstet Gynecol, 1994 Oct, 171(4), 944 - 8
The role of nitric oxide in the pathogenesis of preeclampsia; Seligman SP et al.; OBJECTIVE: Nitric oxide, a potent vasodilator released by endothelial cells, inhibits platelet aggregation and adhesion to vascular endothelial surfaces . Because endothelial cell damage is considered pivotal in the pathogenesis of preeclampsia, this study was initiated to determine whether nitric oxide production is decreased in patients with preeclampsia . STUDY DESIGN: Twenty-six patients with preeclampsia (as defined by a blood pressure > or = 140 mm Hg systolic or 90 mm Hg diastolic plus proteinuria, > or = 300 mg per 24 hours or > or = 2+ by dipstick, both occurring on two occasions > or = 4 hours apart) and 26 normotensive women with singleton gestations in the third trimester were studied . Because nitric oxide is spontaneously oxidized to both nitrite and nitrate, two analytic assays were used serially . Serum nitrite levels were initially determined with the Greiss reagent and subsequently analyzed with Escherichia coli nitrate reductase . RESULTS: With the Greiss reagent alone the mean +/- SEM of serum nitrite level in 26 patients with preeclampsia was significantly decreased compared with 26 normotensive patients (3.46 +/- 1.43 mumol/L vs 4.65 +/- 0.85 mumol/L, p = 0.02) . With the addition of the nitrate reductase enzyme of Escherichia coli the mean +/- SEM of serum nitrite level in 26 preeclamptic patients was again significantly decreased compared with 26 normotensive patients (20.04 +/- 1.25 mumol/L vs 27.38 +/- 2.23 mumol/L, p = 0.02) . One patient with the syndrome of hemolysis, elevated liver enzymes, and low platelets demonstrated a concurrent decrease in serum nitrite over a 2-week period, emphasizing the relationship of nitric oxide to the pathophysiologic features of the syndrome . CONCLUSIONS: Circulating levels of nitrite are decreased in patients with preeclampsia . These data support the concept that diminished nitric oxide synthesis contributes to the pathophysiologic changes seen in preeclampsia.

Scand J Immunol, 1994 Oct, 40(4), 457 - 65
Catabolism of the murine IgG1 molecule: evidence that both CH2-CH3 domain interfaces are required for persistence of IgG1 in the circulation of mice; Kim JK et al.; Site-directed mutagenesis of a recombinant Fc-hinge fragment has previously been used to identify a region of the murine IgG1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains . In the current study the nature of this 'catabolic site' has been further analysed using recombinant techniques . Fc-hinge, CH2-hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice . The CH2-hinge has been analysed as both a monomer and dimer, and the dimer has a longer beta phase half-life (61.6 h) than the monomer (29.1 h) . This suggests that two catabolic sites per Fc fragment are required for serum persistence . The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc-hinge fragment comprising a heterodimer of one Fc-hinge with the wild type (WT) IgG1 sequence and a mutant Fc-hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434) . This hybrid is cleared with a beta phase half-life of 37.9 h and this is significantly shorter than that of the WT Fc-hinge fragment (82.9 h) . In contrast to the CH2-hinge dimer, the CH3 domain is cleared rapidly (beta phase half-life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment . The data extend our earlier observations concerning a region of the murine IgG1 molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.

Mol Immunol, 1994 Oct, 31(14), 1047 - 58
Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies; Kipriyanov SM et al.; A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli . Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster . ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E . coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents . In a final step, the components were separated by size exclusion chromatography . All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis . Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers . ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

Mol Cell Biol, 1994 Oct, 14(10), 6879 - 85
A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions; Maekawa M et al.; We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M . Maekawa, S . Nakamura, and S . Hattori, J . Biol . Chem . 268:22948-22952, 1993) . On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP . The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa . The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene . When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1 . Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene . Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues . A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue . Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype . In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase . These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.

Mol Cell Biol, 1994 Oct, 14(10), 6489 - 96
DNA replication from initiation zones of mammalian cells in a model system; Ishimi Y et al.; We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y . Ishimi and K . Matsumoto, Proc . Natl . Acad . Sci . USA 90:5399-5403, 1993) . In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system . When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase . Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication . The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable . In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication . These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication . The mechanism of initiation is discussed.

J Nucl Med, 1994 Oct, 35(10), 1685 - 90
Synthesis of fluorine-18-labeled biotin derivatives: biodistribution and infection localization; Shoup TM et al.; Recently there has been much interest in the exploitation of the high binding affinity of avidin/biotin as a means of targeting drugs and radionuclides for in vivo applications . We are interested in broadening the application of the avidin/biotin complex to PET . To this end we set out to prepare 18F-labeled biotin analogs . METHODS: Two 18F biotin derivatives, {3aS-(3a alpha,4 beta,6a alpha)}-hexahydro-2-oxo-1H-thieno{3,4- d}imidazole-4-(N-3-(1-{18F}fluoropropyl))pentanamide (1) and {3aS-(3a alpha,4 beta,6a alpha)}-tetrahydro-4-(5-(1-{18F}fluoropentyl)- 1H-thieno{3,4-d}imidazol-2(3H)-(2) were prepared with high specific activity (NCA) and evaluated for their potential in infection localization . RESULTS: Compound 1 binds to avidin and the biodistribution of these derivatives were studied in Escherichia coli infected rats . Half of the infected rats were treated with avidin 24 hr prior to intravenous injection of the 18F-labeled biotin analogs . Biotin 1, without avidin pretreatment, showed a selectivity of 6.08 +/- 1.12 for infection compared to normal muscle . With avidin pretreatment, selectivity increased slightly, giving an infection to normal muscle ratio of 6.39 +/- 0.96 . In contrast, the biodistribution of biotin 2 indicated more binding to normal muscle with an infection to normal muscle ratio of 0.58 +/- 0.07 . This lack of selectivity illustrates the importance of the side-chain amide group in infection localization . There was some defluorination of 1 and 2, as evidenced by increased 18F bone uptake after 60 min: 2.94 +/- 0.37 and 1.17 +/- 0.21 %IG/g +/- s.d., respectively . CONCLUSIONS: Biotin derivatives could be radiofluorinated with high specific activity . Biotin 1, is a potential positron tomography tracer for infection imaging.

J Neurosci, 1994 Oct, 14(10), 5844 - 57
Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles; Masters BA et al.; MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons . The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA . MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum . Transgenic mice generated using 11.5 kb of the mouse MT-III 5' flanking region fused to the E . coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization . MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies . There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles . MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc . These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator.

J Neurol Neurosurg Psychiatry, 1994 Oct, 57(10), 1216 - 20
Mesencephalic and third ventricle cysts: diagnosis and management in four cases; Ramaekers VT et al.; Four infants with obstructive hydrocephalus caused by space occupying third ventricle and mesencephalic cysts are reported . Despite immediate shunt insertion in all patients, there was either lack of clinical improvement or late onset of clinical deterioration . Neuroimaging (CT, MRI, and ventriculography) diagnosed the presence of non-communicating midline outpouchings of the CSF pathways causing obstruction of aqueductal CSF flow and brainstem signs . The cysts were of different origin . In one patient it was caused by a previous thalamic haemorrhage, in another patient by neonatal Escherichia coli meningoventriculitis . In two cases with obstructive hydrocephalus at birth, the aetiology is unclear . Direct puncture and drainage of the cysts led to clinical improvement . The cysts were poorly visualised on CT and could be misinterpreted as an enlarged third ventricle, simulating congenital aqueduct stenosis . Careful neuroradiological investigation is necessary to establish an accurate diagnosis and neurosurgical management . In such cases with hydrocephalus and persisting ventricular enlargement despite shunting, CT ventriculography is a useful tool.

J Gen Virol, 1994 Oct, 75 ( Pt 10), 2803 - 6
Nucleotide sequence of Nilaparvata lugens reovirus genome segment S8 coding for the major outer capsid protein; Nakashima N et al.; The complete nucleotide sequence of genome segment 8 (S8) of Nilaparvata lugens reovirus (NLRV) was determined . It consisted of 1802 nucleotides containing a long open reading frame (562 amino acids), which was expressed in Escherichia coli as a fusion protein . The expressed S8 product, a 62K protein, was detected by Western blotting using IgG directed against intact NLRV particles . This result indicates that S8 encodes the major outer capsid protein of NLRV . The protein exhibited 18.6% amino acid sequence identity with the predicted translation product of S10 of rice black-streaked dwarf virus.

J Gen Virol, 1994 Oct, 75 ( Pt 10), 2761 - 4
The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis; Grierson AW et al.; Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease . Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis . All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease . Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95% . These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.

J Gen Virol, 1994 Oct, 75 ( Pt 10), 2707 - 17
Comparative studies of the structural proteins and glycoproteins of equine herpesviruses 2 and 5; Agius CT et al.; The structural proteins of equine herpesvirus 2 (EHV-2) and EHV-5, recently shown to be gammaherpesviruses, were identified and compared . Labelled proteins and glycoproteins were separated by SDS-PAGE and although EHV-2 and EHV-5 had similar protein profiles, bands in some positions were virus-specific . Six glycoproteins, with distinct profiles, were identified for both EHV-2 and EHV-5 . Rabbit antisera to EHV-2 and EHV-5 and horse antiserum to EHV-2 were used in radioimmunoprecipitations, Western blot analysis and ELISA to investigate the immunogenicity and cross-reactivity of virus proteins . These analyses revealed that while EHV-2 and EHV-5 proteins share many common epitopes, they also possess type-specific epitopes . A 0.71 kb region of the EHV-2 glycoprotein B (gB) gene was expressed as a fusion protein in Escherichia coli . Antiserum raised in a rabbit to the EHV-2 fusion protein was used to identify a 64K EHV-2 protein as EHV-2 gB . Antiserum to EHV-2 gB was used to identify a 66K EHV-5 protein as EHV-5 gB . These proteins, which may represent subunits of gB rather than the entire molecule, appear the most immunodominant of the structural virion proteins as identified by Western blot.

J Gen Virol, 1994 Oct, 75 ( Pt 10), 2567 - 73
Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli; Soumounou Y et al.; The N-terminal P1 protein of turnip mosaic potyvirus (TuMV) polyprotein was overexpressed in Escherichia coli, purified by metal chelation chromatography under denaturing conditions and renatured . U.v . cross-linking experiments indicated that the recombinant protein interacted with RNA, and gel retardation electrophoresis demonstrated that more than one molecule of P1 bound one molecule of RNA . Formation of the protein-RNA complexes was dependent on the conformational state of P1 and was stable at relatively high concentrations of NaCl . P1 had the ability to bind ssRNA and ssDNA, with similar affinity, but was not able to bind to dsDNA . The TuMV protein had the additional characteristic of binding dsRNA with affinity similar to that observed with single-stranded nucleic acids.

J Leukoc Biol, 1994 Oct, 56(4), 453 - 7
Endotoxin stimulates the expression of glucose-6-phosphate dehydrogenase in Kupffer and hepatic endothelial cells; Spolarics Z et al.; The aim of the study was to elucidate the effect of lipopolysaccharide (LPS) administration in vivo (Escherichia coli endotoxin, 1 mg/kg body weight) on the expression and cellular activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), the rate-limiting enzyme of the hexose monophosphate shunt in hepatic cells . Under basal conditions, Kupffer cells displayed higher activity of G6PDH than endothelial or parenchymal cells . In vivo LPS treatments for 7 and 22 h resulted in 40 and 60% increases, respectively, in the cellular activity of G6PDH in Kupffer cells . G6PDH activity was increased by 140 and 90% after 7- and 22-h LPS treatments in endothelial cells . G6PDH activity in parenchymal cells prepared from animals after 22 h of LPS treatment was decreased by approximately 60% compared with that in cells from saline-injected animals . Total cellular RNA or protein extracts from these cells were analyzed by Northern or Western blots . Under basal conditions, G6PDH mRNA levels relative to total cellular RNA were higher in Kupffer than in endothelial cells and were not detectable in parenchyma cells . LPS injection caused a time-dependent increase in G6PDH mRNA expression in Kupffer and endothelial cells . Western blot analysis of Kupffer cell extracts also showed that LPS treatments caused markedly elevated expression of protein in these cells . These results show that endotoxemia results in marked induction of G6PDH in Kupffer and hepatic endothelial cells but has no such effect in the parenchymal cells . These findings also suggest that the elevated cellular expression of G6PDH is an important regulatory event in the adaptive responses of hepatic nonparenchymal cells to infections . The elevated expression of G6PDH may be important for support of the upregulated NADPH-dependent pathways, such as superoxide anion and nitric oxide production, macromolecular synthesis, or the maintenance of cellular glutathione status.

J Infect Dis, 1994 Oct, 170(4), 841 - 7
Reduced virulence of rifampicin-resistant mutants of Francisella tularensis; Bhatnagar N et al.; Rifampicin-resistant mutants of a live vaccine strain (LVS) of Francisella tularensis were produced and screened for virulence in mice; 4 avirulent clones with intraperitoneal (ip) LD50s > 10(6) cfu, compared with 10(2) cfu for LVS, were characterized . Growth characteristics at 37 degrees C, surface envelope proteins, and lipopolysaccharide profiles of resistant mutants were identical to those of LVS . Polymerase activity of the mutants was more resistant than the enzyme from LVS to the inhibitory action of rifampicin . Growth rates for mutants and LVS were similar during the first 5 h at 42 degrees C, but viability of the mutants decreased to < 0.01% at 24 h . LVS and mutants differed in their ability to grow in vitro in host macrophages: LVS increased 580-fold over 72 h; mutants increased 33-fold . After ip inoculation of the organisms into mice, increasing numbers of LVS from peritoneal cells were isolated; mutants decreased over 4 days.

J Allergy Clin Immunol, 1994 Oct, 94(4), 689 - 98
Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species; Laffer S et al.; BACKGROUND: Grass pollens, such as pollen from timothy grass (Phleum pratense), represent a major cause of type I allergy . OBJECTIVE: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens . METHODS: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergen Phl p I . A recombinant Phl p I-beta-galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli . Using recombinant Phl p V and Phl p I, we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens . IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution . RESULTS: Preadsorption of patients' sera with recombinant nonfusion Phl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species . CONCLUSION: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species.

J Cell Physiol, 1994 Oct, 161(1), 149 - 59
Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF; Gualandris A et al.; The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M(r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA . As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells . To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon . Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein . When the same 24-kD bFGF cDNA was expressed in E . coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography . Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells . In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea . Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures . Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF . Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment . At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products . In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo . Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells.

J Cell Biol, 1994 Oct, 127(2), 467 - 78
Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase); Tassan JP et al.; The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2 . One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish . It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit . In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle . Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity . We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli . These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle . Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15 . The association of the three proteins is near stoichiometric and invariant throughout the cell cycle . Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis . The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise . It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

J Bacteriol, 1994 Oct, 176(20), 6404 - 6
Additive effect of tolC and rfa mutations on the hydrophobic barrier of the outer membrane of Escherichia coli K-12; Fralick JA et al.; Studies using tolC mutant derivatives of deep rough (rfa) mutants indicate that tolC and rfa mutations have an additive effect with respect to their sensitivity to hydrophobic agents, suggesting that they are not acting through a mutual mechanism to alter the permeability of the outer membrane.

J Bacteriol, 1994 Oct, 176(20), 6392 - 6
Maturation and localization of the TolB protein required for colicin import; Isnard M et al.; The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB*) . To explain the presence of these two forms, two hypotheses have been proposed: TolB might be posttranslationally processed to TolB*, or an internal in-frame translation initiation resulting in TolB* may occur (S . K . Levengood and R . E . Webster, J . Bacteriol . 171:6600-6609, 1989) . To address this question, TolB was tagged by inserting in its C-terminal region an epitope recognized by monoclonal antibody 1C11 without altering the function of TolB . It was then demonstrated that the functional protein corresponded to TolB*, the mature periplasmic protein, and that TolB was its precursor form, which was observed only when the protein was overexpressed . These two forms were purified by immunoprecipitation, and their N-terminal sequences were determined . An antibody directed against TolB was raised, which confirmed the results obtained with the tagged TolB.

J Bacteriol, 1994 Oct, 176(20), 6340 - 8
Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor; Ames P et al.; Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes . One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation . To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells . Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal . The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis . Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor . CheA was required for both signaling activities, whereas CheW was needed only for CW signaling . Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro . Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA . CheW was required for stimulation but not for inhibition . These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals . The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well . The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.

J Bacteriol, 1994 Oct, 176(20), 6255 - 61
The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity; Chen BJ et al.; Escherichia coli can ameliorate the toxic effects of alkylating agents either by preventing DNA alkylation or by repairing DNA alkylation damage . The alkylation-sensitive phenotype of E . coli alkB mutants marks the alkB pathway as an extremely effective defense mechanism against the cytotoxic effects of the SN2, but not the SN1, alkylating agents . Although it is clear that AlkB helps cells to better handle alkylated DNA, no DNA alkylation repair function could be assigned to the purified AlkB protein, suggesting that AlkB either acts as part of a complex or acts to regulate the expression of other genes whose products are directly responsible for alkylation resistance . However, here we present evidence that the provision of alkylation resistance is an intrinsic function of the AlkB protein per se . We expressed the E . coli AlkB protein in two human cell lines and found that it confers the same characteristic alkylation-resistant phenotype in this foreign environment as it does in E . coli . AlkB expression rendered human cells extremely resistant to cell killing by the SN2 but not the SN1 alkylating agents but did not affect the ability of dimethyl sulfate (an SN2 agent) to alkylate the genome . We infer that SN2 agents produce a class of DNA damage that is not efficiently produced by SN1 agents and that AlkB somehow prevents this damage from killing the cell.

J Bacteriol, 1994 Oct, 176(20), 6245 - 54
Role of regulatory features of the trp operon of Escherichia coli in mediating a response to a nutritional shift; Yanofsky C et al.; Physiological studies were performed under nutritional stress and nonstress conditions to assess the relative importance of the various regulatory mechanisms that Escherichia coli can use to alter its rate of tryptophan synthesis . Mutants were examined in which the trp repressor was inactive, transcription termination at the trp attenuator was altered, transcription initiation at the trp promoter was reduced, or feedback inhibition of anthranilate synthase was abolished . Strains were examined in media with and without tryptophan, phenylalanine and tyrosine, or acid-hydrolyzed casein and following shifts from one medium to another . Growth rates and anthranilate synthase levels were measured . In media lacking tryptophan, each of the mutants showed relief of repression and/or attenuation and maintained a near-normal growth rate . Following a shift from a medium containing tryptophan to a tryptophan-free medium containing phenylalanine and tyrosine or acid-hydrolyzed casein, mutants with abnormally low trp enzyme levels exhibited an appreciable growth lag before resuming growth . The wild-type strain displayed termination relief only under one extreme shift condition, upon transfer from a minimal medium containing tryptophan to minimal medium with only phenylalanine and tyrosine . A promoter down-mutant had difficulty adjusting to a shift from high tryptophan to low tryptophan levels in a medium containing acid-hydrolyzed casein . In all media tested, anthranilate synthase levels were lower in a feedback-resistant mutant than in the wild type . These studies demonstrate the capacity of E . coli to adjust its rate of tryptophan synthesis to maintain rapid growth following a shift to stressful nutritional conditions.

J Bacteriol, 1994 Oct, 176(20), 6214 - 20
Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR; Hagewood BT et al.; The efficient expression of the tdc operon of Escherichia coli requires the products of two regulatory genes, tdcR and tdcA . We have identified the transcription site of tdcR by primer extension mapping and established the translation start site of TdcR by mutational analysis of its reading frame . In a tdcR tdcABC deletion strain, tdcR+ promoted high-level LacZ expression from a lambda tdcAB-lacZ lysogen and mutations introduced in tdcR resulted in a greater than sixfold decrease in LacZ level . In-frame deletions of tdcA also reduced LacZ expression, and chromosomal and plasmid-borne tdcA+ increased the LacZ level in tdcA mutant lysogens . Interestingly, multicopy tdcA+ plasmids introduced into tdcR mutant strains completely restored tdc expression . In separate experiments we found that mutations in the tdc promoter DNA around positions -70, -140, and -175 greatly reduced tdc expression relative to that for the wild-type promoter and the tdcP mutation around -175 prevented multicopy tdcA+ from rescuing tdcR mutants . Furthermore, competition experiments revealed that a wild-type promoter fragment encompassing the -175 region cloned into a plasmid reduced tdc expression by titrating TdcA in vivo, and this effect was reversed with excess TdcA . These results suggest that in tdcR+ cells TdcR interacts with tdcP and/or TdcA to enhance tdc transcription whereas in tdcR mutant cells a new tdcP-TdcA complex around -175 in the native promoter bypasses the requirement for TdcR . On the basis of the accumulated data summarized here and elsewhere we propose that multiple transcription factors enhance tdc operon expression by bending and looping of the promoter DNA to form an active transcription complex.

J Bacteriol, 1994 Oct, 176(20), 6159 - 64
Characterization of the gcv control region from Escherichia coli; Stauffer LT et al.; We constructed a set of deletions upstream of the gcv promoter and analyzed the effects of the deletions on expression of a gcvT-lacZ gene fusion . A deletion that ends at position -313 upstream of the transcription initiation site (+1) results in reduced levels of gcvT-lacZ expression, but the fusion is still inducible by glycine and repressible by purines . A deletion that ends at position -169 results in loss of both GcvA- and Lrp-mediated activation of the gcvT-lacZ fusion . The endpoints of delta -313 and delta -169 also define a site that down-regulates gcvT-lacZ expression two- to threefold . A deletion that ends at position -89 upstream from the transcription initiation site still shows PurR-mediated repression, suggesting that PurR-mediated repression is not by direct interference with the GcvA- and Lrp-mediated regulatory mechanism(s) . Gel mobility shift assays and DNase I footprinting showed that Lrp protein binds to multiple sites upstream of the gcv promoter, from about bp -92 to bp -229 . The results suggest that the gcv regulatory region is complex, with numerous cis-acting sites that are required for normal gcv expression.

J Bacteriol, 1994 Oct, 176(19), 6143 - 5
The min locus, which confers topological specificity to cell division, is not involved in its coupling with nucleoid separation; Dassain M et al.; In Escherichia coli, nucleoid separation and cell constriction remain tightly linked when division is retarded by altering the level of synthesis of the protein FtsZ . In this study, we have examined the role of the min locus, which is responsible for the inactivation of polar division sites, in the partition-septation coupling mechanism . We conclude that the coupling persists in a delta min strain and that its timing relative to replication remains dependent on the level of FtsZ synthesis . We suggest that the retarded nucleoid segregation observed in min mutants is the result of this coupling in cells with a perturbed pattern of nonpolar divisions.

J Bacteriol, 1994 Oct, 176(19), 6134 - 8
An Escherichia coli K-12 tktA tktB mutant deficient in transketolase activity requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth; Zhao G et al.; We show that a tktA tktB double mutant, which is devoid of the two known transketolase isoenzymes of Escherichia coli K-12, requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth . This pyridoxine requirement can also be satisfied by 4-hydroxy-L-threonine or glycolaldehyde . These results provide direct evidence that D-erythrose-4-phosphate is a precursor of the pyridine ring of pyridoxine . In addition, they show that the two major E . coli transketolase isoenzymes are not required for the biosynthesis of D-1-deoxyxylulose, which is thought to be another precursor of pyridoxine.

J Bacteriol, 1994 Oct, 176(19), 6100 - 6
Relationship between ftsZ gene expression and chromosome replication in Escherichia coli; Zhou P et al.; Transcriptional levels within the ftsQAZ region of the Escherichia coli chromosome were correlated with chromosome replication and the division cycle . The transcripts were measured either in synchronous cultures generated by the baby machine technique or in dnaC2(Ts) mutants that had been aligned for initiation of chromosome replication by temperature shifts . Transcription within the ftsZ reading frame was found to fluctuate during the cell cycle, with maximal levels about midcycle and a minimum level at division, in cells growing with a doubling time of 24 min at 37 degrees C . Examination of transcription in dnaC(Ts) mutants aligned for chromosome replication indicated that the periodicity was due to a reduction in transcripts coincident with replication of the ftsQAZ region . Transcription originating upstream of the ftsA gene exhibited the periodicity and accounted for a significant proportion of the transcripts entering ftsZ . The most obvious interpretation of the data is that replication of the region transiently inhibits transcription, but alternative explanations have not been ruled out . However, no other relationship between transcription and either replication or division was detected.

J Bacteriol, 1994 Oct, 176(19), 6039 - 44
DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect; Williams SG et al.; The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed . However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains . The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur . A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene . A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication . DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A . In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.

J Bacteriol, 1994 Oct, 176(19), 6015 - 22
A new gene involved in stationary-phase survival located at 59 minutes on the Escherichia coli chromosome; Li C et al.; We determined the DNA sequence of a 2,232-bp region immediately upstream of the pcm gene at 59 min on the Escherichia coli chromosome that encodes an L-isoaspartyl protein methyltransferase with an important role in stationary-phase survival . Two open reading frames of 477 and 1,524 bp were found oriented in the same direction as that of the pcm gene . The latter open reading frame overlapped the 5' end of the pcm gene by 4 bp . Coupled in vitro transcription-translation analysis of DNA containing the 1,524-bp open reading frame directly demonstrated the production of a 37,000-Da polypeptide corresponding to a RNA species generated from a promoter within the open reading frame . The deduced amino acid sequence showed no similarity to known protein sequences . To test the function of this gene product, we constructed a mutant strain in which a kanamycin resistance element was inserted at a BstEII site in the middle of its coding region in an orientation that does not result in reduction of Pcm methyltransferase activity . These cells were found to survive poorly in stationary phase, at elevated temperatures, and in high-salt media compared with parent cells containing the intact gene, and we thus designate this gene surE (survival) . surE appears to be the first gene of a bicistronic operon also containing the pcm gene . The phenotypes of mutations in either gene are very similar and indicate that both gene products are important for the viability of E . coli cells under stressful conditions.

J Bacteriol, 1994 Oct, 176(19), 5958 - 61
Conjugal mobilization of plasmid DNA: termination frequency at the origin of transfer of plasmid R1162; Rao XM et al.; Conjugal transfer of plasmid R1162 is initiated and terminated at a 38-bp origin of transfer (oriT) . Plasmids containing two directly repeated copies of oriT were used to determine the actual frequency of termination at this site . This frequency was calculated both for oriTnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated oriT where transfer had been initiated . In both cases, the termination frequency decreased as the distance between the initiation and termination sites became greater and was significantly less than one for plasmids the size of R1162 . A substantial proportion of recipient cells received more than one plasmid copy during transfer . Our results indicate that termination is inefficient but that this is partly compensated for by the transmission of multiple plasmid copies.

J Bacteriol, 1994 Oct, 176(19), 5938 - 48
Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori; Kostrzynska M et al.; Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000 . The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving . The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry . The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H . pylori promoter . lpp20 was mapped to the same 358-kb NruI fragment as flaB . DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site . The mass of the deduced 154-residue mature protein was 16,865 Da . Growth of E . coli cells expressing the cloned H . pylori lpp20 gene in the presence of {3H}palmitic acid resulted in radiolabelled Lpp20 while treatment of the E . coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein . Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane . A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

J Bacteriol, 1994 Oct, 176(19), 5888 - 96
Response to UV damage by four Escherichia coli K-12 restriction systems; Kelleher JE et al.; To understand the role of restriction in regulating gene flow in bacterial populations, we would like to understand the regulation of restriction enzyme activity . Several antirestriction (restriction alleviation) systems are known that reduce the activity of type I restriction enzymes like EcoKI in vivo . Most of these do not act on type II or type III enzymes, but little information is available for the unclassified modification-dependent systems, of which there are three in E . coli K-12 . Of particular interest are two physiological controls on type I enzymes: EcoKI restriction is reduced 2 to 3 orders of magnitude following DNA damage, and a similar effect is seen constitutively in Dam- cells . We used the behavior of EcoKI as a control for testing the response to UV treatment of the three endogenous modification-dependent restriction systems of K-12, McrA, McrBC, and Mrr . Two of these were also tested for response to Dam status . We find that all four resident restriction systems show reduced activity following UV treatment, but not in a unified fashion; each response was genetically and physiologically distinct . Possible mechanisms are discussed.

Infect Immun, 1994 Oct, 62(10), 4686 - 9
Analysis of a naturally occurring K99+ enterotoxigenic Escherichia coli strain that fails to produce K99; Isaacson RE et al.; A spontaneously occurring field isolate of enterotoxigenic Escherichia coli that was genotypically K99+ but phenotypically K99- was analyzed for the reason that it did not express K99 . The defect, which was cis active, was located within an area 5' to the first gene required for K99 biogenesis and was the result of the deletion of a single base pair.

Infect Immun, 1994 Oct, 62(10), 4632 - 6
Biological activities of native and recombinant Borrelia burgdorferi outer surface protein A: dependence on lipid modification; Weis JJ et al.; Borrelia burgdorferi lipoproteins are 50- to 500-fold more active as cytokine inducers and B-cell mitogens than Escherichia coli lipoproteins and synthetic peptides containing the tripalmitoyl-S-glyceryl-cysteine moiety . To investigate the source of this unique potency, we compared native OspA from B . burgdorferi with recombinant lipidated OspA produced in E . coli . As little as 10 ng of either protein per ml stimulated B-cell proliferation and production of cytokines and nitric oxide by macrophages . The two proteins induced comparable antibody responses in mice . Nonlipidated OspA made in E . coli had no stimulatory activity . Thus, lipid modification is essential both in vivo and in vitro for the immunological properties of OspA . The lipid moiety appears equally active whether produced in B . burgdorferi or in E . coli.

Infect Immun, 1994 Oct, 62(10), 4594 - 601
Putative adhesins of Anaplasma marginale: major surface polypeptides 1a and 1b; McGarey DJ et al.; Genes for the MSP1a and MSP1b subunits of the Anaplasma marginale surface antigen complex MSP1 were previously cloned and expressed in Escherichia coli . We report here the localization of MSP1a and MSP1b polypeptides on the surface of recombinant E . coli by using a live cell indirect immunofluorescent antibody assay . Recombinant E . coli cells expressing the msp1 alpha gene or the msp1 beta gene encoding the MSP1a and MSP1b polypeptide subunits, respectively, were shown by a culture recovery adhesion assay and by direct microscopic examination to specifically adhere to bovine erythrocytes . This adhesion was more than additive when both genes were coexpressed in a single recombinant construct . Similarly, these recombinants hemagglutinated bovine erythrocytes in a microtiter hemagglutination assay . Inhibition of recombinant E . coli adhesion to bovine erythrocytes and hemagglutination inhibition were observed in the presence of homologous monospecific polyclonal antiserum raised against purified MSP1a or MSP1b polypeptide . These data suggest that the MSP1a and MSP1b polypeptides have functions as adhesins on A . marginale initial bodies, probably during erythrocyte invasion.

Infect Immun, 1994 Oct, 62(10), 4411 - 8
Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons; Jansen R et al.; Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A . pleuropneumoniae, the causative agent of porcine pleuropneumonia . Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized . The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A . pleuropneumoniae, i.e., ApxI and ApxII . In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8 . This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively . Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB . We examined the presence of apxIIICABD sequences in the 12 serotypes of A . pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII . Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences . Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes . The differences were located between the glycine-rich repeats and the secretion signal . The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A . pleuropneumoniae . We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.

Infect Immun, 1994 Oct, 62(10), 4339 - 46
Monoclonal antibodies against enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) that cross-react immunologically with heterologous CFAs; Rudin A et al.; Enterotoxigenic Escherichia coli binds to enterocytes in the small intestine by means of antigenically distinct colonization factors (CFs), usually termed colonization factor antigens (CFAs), coli surface antigens (CS), or putative colonization factor antigens (PCFs) . To explore the immunological relationship between different CFs, we dissociated CFA/I fimbriae into subunits and produced monoclonal antibodies (MAbs) against these subunits . We selected three MAbs that cross-reacted immunologically with a number of different, whole purified CFs in a dot blot test and with the corresponding subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . One of the MAbs, i.e., subunit CFA/I 17:8 (S-CFA/I 17:8), reacted more strongly with subunits of CFA/I than with whole purified fimbriae . This MAb cross-reacted with whole purified fimbriae and subunits of CS4, PCFO166, CS1, and CS2 . Moreover, it bound strongly to a peptide of 25 amino acids corresponding to the N-terminal end of CFA/I . The other two MAbs, i.e., S-CFA/I 5:6 and S-CFA/I 8:11, cross-reacted with CS1, CS2, CS4, PCFO166, and CS17 fimbriae but reacted only slightly or not at all with the CFA/I peptide . MAbs S-CFA/I 17:8 and S-CFA/I 5:6 were shown to inhibit hemagglutination by bacterial strains that express either CFA/I, CS1, or CS4 . In addition, the binding of enterotoxigenic E . coli strains expressing CFA/I, CS2, CS4, and PCFO166 to enterocyte-like cell-line Caco-2 was inhibited by both MAbs . These results show that several antigenically different CFs have common epitopes and that among these at least one is located in the N-terminal end of the subunit protein . Moreover, antibodies against the common epitopes seem to block binding of the bacterial strains that express different CFs to both erythrocytes and Caco-2 cells.

Infect Immun, 1994 Oct, 62(10), 4270 - 8
Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin; Grant CC et al.; Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity . Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity . To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined . The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis . The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E . coli . A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells . The results indicate that trypsin-like cleavage of the A subunit of E . coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects.

Infect Immun, 1994 Oct, 62(10), 4233 - 43
A minor 987P protein different from the structural fimbrial subunit is the adhesin; Khan AS et al.; The 987P fimbriae produced by enterotoxigenic strains of Escherichia coli isolated from piglets mediate bacterial attachment to intestinal epithelial cells . These fimbriae consist essentially of a tight helical arrangement of one structural protein subunit encoded by fasA . Fimbriation and specific adhesion requires the expression of seven additional genes (fasB to fasH) . In this study, we investigated whether FasA or another Fas protein, e.g., a potential minor fimbrial component, harbors the binding moiety for the pig 987P receptor glycoproteins . Fas proteins, specifically radiolabeled with an in vivo T7 expression system, were isolated from the periplasm and incubated with receptor-containing brush borders isolated from piglet intestinal epithelial cells . FasG bound best to brush borders, whereas no FasA adhered to them . Additional evidence that FasG, and not FasA, is the 987P adhesin was provided by ligand blotting inhibition assays indicating that FasG alone inhibited fimbrial binding to 987P receptors and that in the absence of FasG, other Fas proteins were not inhibitory . FasG was identified in purified fimbrial preparations with a specific anti-FasG antibody probe . Moreover, FasG was shown to be tightly associated with the fimbrial structure, since it was released only after disassembling fimbriae by heat and sodium dodecyl sulfate treatments . The primary structure of FasG, deduced from the DNA sequence, exhibited 19.1 to 24.4% similarity to FasA and large minor components and/or adhesins of other fimbriae . FasG is the first-described minor fimbrial subunit shown to be essential for both fimbrial biogenesis and specific adhesion.

Infect Immun, 1994 Oct, 62(10), 4208 - 18
Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli; Sirakova TD et al.; An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis and patient/and an internal peptide derived from it were subjected to N-terminal sequencing . Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme . The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame . According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase . This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide . A . fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases . The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme . Although the enzyme in the inclusion bodies could not be renatured, expression at 30 degrees C yielded soluble enzyme that showed chromatographic behavior identical to that of the native fungal enzyme and catalyzed hydrolysis of elastin . The metalloproteinase gene described here was not found in Aspergillus flavus.

Infect Immun, 1994 Oct, 62(10), 4153 - 9
Virulence properties and attaching-effacing activity of Escherichia coli O45 from swine postweaning diarrhea; Zhu C et al.; Escherichia coli O45 isolates associated with swine postweaning diarrhea in Quebec were characterized with respect to virulence determinants genetically and investigated for their attaching and effacing (A/E) activities by experimental inoculation of gnotobiotic piglets and by the HEp-2 cell adherence assay . All of 32 isolates tested were negative for enterotoxigenic and verotoxigenic E . coli virulence determinants, heat-labile enterotoxin (LT), heat-stable enterotoxins (STap, STb), verotoxins (VT1, VT2), and F4 (K88), F5 (K99), F6 (987P), and F41, except one STb-positive and two F4-positive isolates . A total of 25 isolates hybridized with an EaeA probe, and 11 hybridized with an enteropathogenic E . coli adherence factor (EAF) probe . None of 32 isolates hybridized with a bundle-forming pilus (BFP) probe . The EAF, EaeA, and BFP factors have been associated with human enteropathogenic E . coli strains . A total of 10 of 12 eaeA-positive porcine O45 isolates induced A/E lesions characterized by intimate adherence of bacteria to the intestinal epithelial cell membrane with effacement of the microvilli, similar to those of human attaching-effacing E . coli . However, A/E lesions were not observed in the piglets inoculated with any one of three eaeA-negative O45 isolates . All E . coli O45 isolates were non-adherent to HEp-2 cells . Thus, we have demonstrated the production of typical A/E lesions by nonenterotoxigenic E . coli O45 isolates from swine postweaning diarrhea . The results indicate the significance of the eaeA gene in A/E activities of these isolates and suggest that EAF and BFP are not involved in O45 E . coli infection of weaning piglets.

Infect Immun, 1994 Oct, 62(10), 4124 - 34
Effects of temperature, time, and toxin concentration on lesion formation by the Escherichia coli hemolysin; Moayeri M et al.; We performed osmotic protection experiments to test the hypothesis that the Escherichia coli hemolysin forms a discrete-size pore in erythrocyte membranes . The effects of toxin concentration, assay time, temperature, and protectant concentrations were examined . The results we present here raise doubts about the existing model of pore formation by hemolysin . We demonstrate that osmotic protection by various sugars of different sizes is a function of hemolysin concentration and assay time . The data indicate that under various conditions, lesion sizes with a diameter ranging from < 0.6 to > 1.2 nm can be inferred . Quantification of hemolysin permitted the estimation of the number of HlyA structural protein molecules required per erythrocyte for lysis in the presence of each protectant . It appears that hemolysin induces heterogeneous erythrocyte lesions which increase in size over time . Influx experiments utilizing radioactive sugar markers indicated that time-dependent osmotic protection patterns are independent of the diffusion rates of individual protectants . We demonstrate that the rate of the putative growth in the size of hemolysin-mediated lesions is temperature dependent . The erythrocyte membrane lesions formed at 37 degrees C can be stabilized in size when shifted to 4 degrees C . On the basis of these data, new models for the nature of the hemolysin-mediated erythrocyte membrane lesions are presented.

Gastroenterology, 1994 Oct, 107(4), 950 - 6
Escherichia coli heat-stable enterotoxin-mediated colonic Cl- secretion is absent in cystic fibrosis; Goldstein JL et al.; BACKGROUND/AIMS: Calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretions in the human colon are abnormal in cystic fibrosis, but the effect of guanosine 3',5'-cyclic monophosphate (cGMP) is unknown . This study examined the effects of the cGMP activator Escherichia coli heat-stable enterotoxin (STa) on rectal ion transport of controls and subjects with cystic fibrosis . METHODS: In vivo rectal potential difference (PD) was measured in response to 10(-7) mol/L STa in adult cystic fibrosis (n = 6) and control subjects (n = 7) . Cl- transport was also evaluated in 24-hour primary cultures of human colonocytes using 6-methoxy-quinolyl-acetoethyl ester in response to STa (1 mumol/L) and 8-bromo-cGMP (100 mumol/L) with or without Cl- transport inhibitors . RESULTS: Whereas STa increased rectal potential difference in controls, there was no effect in cystic fibrosis subjects . STa stimulated the cGMP concentration in rectal biopsy specimens from both control and cystic fibrosis subjects approximately twofold . In vitro Cl- transport in non-cystic fibrosis colonocytes increased threefold and fivefold with STa and 8-bromo-cGMP, respectively . These transport increases were inhibited by furosemide and the Cl- channel blocker diphenylamine-2-carboxylate . CONCLUSIONS: Human colonocytes secrete Cl- in response to STa and cGMP in normal subjects, but this response is absent in cystic fibrosis.

Gastroenterology, 1994 Oct, 107(4), 1075 - 84
Decreased bilirubin transport in the perfused liver of endotoxemic rats; Roelofsen H et al.; BACKGROUND/AIMS: Hyperbilirubinemia associated with sepsis is frequently observed in humans . In this study, an experimental rat model was developed to study bilirubin metabolism and transport during endotoxemia . METHODS: Rats were injected intravenously with a single bolus of lipopolysaccharide (1 mg/kg); after 18 hours, the liver was removed for single-pass perfusion . Unconjugated bilirubin, bilirubin ditaurate (125 nmol/min), and/or taurocholate (1.5 mumol/min) were infused . Rate constants for uptake were determined from the disappearance of a bolus of bilirubin ditaurate in a recirculating perfusion . RESULTS: In endotoxemic livers, biliary excretion of bilirubin-glucuronides was reduced by 49% (2.04 +/- 0.2 and 3.99 +/- 0.24 nmol.min-1.g liver-1) . Similar results were obtained with bilirubin ditaurate, indicating that the reduced transport is not caused by a reduced conjugation capacity . The rate constant of sinusoidal uptake was significantly reduced during endotoxemia (0.191 +/- 0.034 vs . 0.090 +/- 0.035, respectively) . Secretion of taurocholate into bile was also reduced (92 +/- 22 vs . 127 +/- 10 nmol.min-1.g liver-1) . CONCLUSIONS: In endotoxemic rats, biliary clearance of bilirubin and taurocholate is substantially decreased, suggesting that decreased output of bilirubin-glucuronides is not caused by impaired conjugation but by a reduction in transport.

Exp Cell Res, 1994 Oct, 214(2), 595 - 605
Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells; Solberg R et al.; The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase . Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin . The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum . Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha . The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP . The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta . Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP . The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm . A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus . When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types . REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme . In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.

Eur J Biochem, 1994 Oct 1, 225(1), 99 - 105
GTP-blot analysis of small GTP-binding proteins . The C-terminus is involved in renaturation of blotted proteins; Klinz FJ; Recombinant c-Ha-ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind {alpha-32P}GTP after SDS/PAGE and transfer to nitrocellulose . Recombinant c-Has-ras missing the C-terminal 23 amino acid residues failed to bind {alpha-32P}GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind {alpha-32P}GTP was decreased many-fold . The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective . We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process . Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of {alpha-32P}GTP as expected from the level of ras immunoreactivity . Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol . Examination of {alpha-32P}GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of {alpha-32P}GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras . Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.

Eur J Biochem, 1994 Oct 1, 225(1), 491 - 9
Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.); Appert C et al.; Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petroselium crispum Nym.) were cloned and the complete amino acid sequences deduced . Fusion proteins with glutathione S-transferase were expressed in Escherichia coli, purified and cleaved . All of the resulting phenylalanine ammonia-lyase proteins, as well as the fusion proteins, were catalytically active . The turnover number of one selected isoenzyme, PAL-1, was estimated to be around 22 s-1 for each active site . In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia-lyase isoenzymes exhibited very similar apparent Km values for L-phenylalanine (15-24.5 microM) as well as identical temperature (58 degrees C) and pH (8.5) optima . All of them were competitively inhibited by (E)-cinnamate with similar efficiency (Ki values: 9.1-21.5 microM), lacked cooperative behaviour, and accepted L-tyrosine as a substrate with low affinity (Km values: 2.6-7.8 mM) . These results suggest that the occurrence of multiple gene copies has a function other than encoding isoenzymes with different enzyme kinetic properties.

Eur J Biochem, 1994 Oct 1, 225(1), 449 - 57
Purification and characterisation of recombinant sea urchin metallothionein expressed in Escherichia coli; Wang Y et al.; Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururatus . To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium-containing form, from synthetic cDNA heterologously expressed in Escherichia coli . The purified protein was identified as the desired product by a combination of peptide-map analysis, amino acid sequence analysis and ion-spray mass spectroscopy . The existence of seven 113Cd NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule . The position of the NMR resonances (605-695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmium-thiolate clusters . With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape . Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal-thiolate clusters of distinctly different stability . At pH 7 the average apparent association constant for Cd2+ in the clusters is about 20-times weaker in sea urchin MTA than in rabbit MT-2.

Eur J Biochem, 1994 Oct 1, 225(1), 43 - 51
Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase; Veiga-Da-Cunha M et al.; Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate {Vandercammen A . & Van Schaftingen, E . (1993) Biochem . J . 294, 551-556} . The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart . A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe . The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein . This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues . The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver . In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase . These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.

Eur J Biochem, 1994 Oct 1, 225(1), 411 - 7
Comparison of the inhibitory action of natural rotenone and its stereoisomers with various NADH-ubiquinone reductases; Ueno H et al.; Two stereoisomers of natural rotenone (5'alpha-epirotenone and 5'beta-epirotenone) were synthesized to identify the stereochemical factor of rotenone required for the inhibition and also to probe the structure of the rotenone binding site . The inhibitory action of the stereoisomers was compared with that of rotenone using NADH-ubiquinone reductases from bovine heart submitochondrial particles (SMP), potato tubers (Solanum tuberosum L.) SMP and Escherichia coli (GR19N) membranes . With respect to bovine heart SMP, it was found that the bent form of rotenone is essential for the activity . The modification of the E-ring moiety also affected both the inhibitory potency and the pattern of inhibition . These results indicated that the rotenone-binding site recognizes the whole molecular structure (or shape) of rotenone in a strict sense . Rotenone and 5'beta-epirotenone inhibited the NADH-ubiquinone reductase of bovine heart SMP in a noncompetitive manner against exogenous quinones . In contrast, the inhibition pattern of 5'alpha-epirotenone varied from noncompetitive to competitive as the concentration of quinone increased . These results suggest that rotenone binds close to, but not at a site identical to, the location for ubiquinone in the ubiquinone-catalytic reaction site, whereas the 5'alpha-epirotenone-binding site overlaps that for ubiquinone due to a structural modification of E-ring moiety . Furthermore, the complex inhibition pattern of 5'alpha-epirotenone suggests that there are two quinone-binding sites in NADH-ubiquinone reductase . In contrast, the order of the inhibitory potencies of the three inhibitors with proton-pumping NADH-ubiquinone reductase of potato SMP was the same as that observed for the bovine enzyme . This suggests that the structure of rotenone-binding sites (or ubiquinone-binding sites) of these enzymes are similar . It was further demonstrated that 5'alpha-epirotenone inhibits quinone binding to both proton-pumping and non-proton-pumping NADH-ubiquinone reductases of potato SMP in a competitive manner . With respect to the proton-pumping NADH-ubiquinone reductase of the E . coli membrane, the sensitivity of the enzyme to the inhibitor was remarkably decreased and the difference in the inhibitory potencies of the three inhibitors became ambiguous . In addition, the inhibition pattern of the three inhibitors was competitive against quinone . These results indicated that, contrary to the mammalian enzyme, only part of the rotenone molecule is recognized by the quinone-binding site of this enzyme.

Eur J Biochem, 1994 Oct 1, 225(1), 395 - 401
The function of arginine 363 as the substrate carboxyl-binding site in Escherichia coli serine hydroxymethyltransferase; Delle Fratte S et al.; Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues . Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme-bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs . The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild-type enzyme . The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild-type enzyme and a 15-fold reduction in affinity for glycine and serine . The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate . Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro-aldol cleavage of serine ethyl ester and serinamide . The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild-type enzyme activity with serine, respectively . The R363A mutant enzyme catalyzed the half transamination of D-alanine methyl ester and L-alanine methyl ester at rates similar to the rates of transamination of D-alanine and L-alanine by the wild-type enzyme . The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase . Evidence is also provided that R363 may play a role in the substrate-induced open to closed conformational change of the active site.

Eur J Biochem, 1994 Oct 1, 225(1), 321 - 31
Multiple states of the molybdenum centre of dimethylsulphoxide reductase from Rhodobacter capsulatus revealed by EPR spectroscopy; Bennett B et al.; The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group . Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical . EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals . These could be simulated, indicating that each corresponds to a single chemical species . Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature . Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand . One signal with gav 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with gav values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli . These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor . Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both . The latter may involve replacement of an amino acid residue co-ordinating molybdenum via O or N, for a cysteine co-ordinating via S . Since the two signals with gav 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.

Eur J Biochem, 1994 Oct 1, 225(1), 253 - 61
The heterodisulfide reductase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic of pyridine-nucleotide-dependent thioredoxin reductases; Hedderich R et al.; The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron-sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced . HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD-binding domain of pyridine-nucleotide-dependent disulfide oxidoreductases . Additionally, 110 amino acids C-terminal to the FAD-binding consensus, a short polypeptide stretch (VX2CATID) was detected which shows similarity to the region of thioredoxine reductase that contains the active-site cysteine residues (VX2CATCD) . These findings suggest that HdrA harbors the site of heterodisulfide reduction and that the catalytic mechanism of the enzyme is similar to that of pyridine-nucleotide-dependent thioredoxin reductase . HdrA was additionally found to contain four copies of the sequence motif CX2CX2CX3C(P), indicating the presence of four {4Fe-4S} clusters . Two such sequence motifs were also present in HdrC (21.76 kDa), the N-terminal amino acid sequence of which showed sequence similarity to the gamma-subunit of the anaerobic glycerol-3-phosphate dehydrogenase of Escherichia coli . HdrC is therefore considered to be an electron carrier protein that contains two {4Fe-4S} clusters . HdrB (33.46 kDa) did not show sequence similarity to other known proteins, but appears to possess a C-terminal hydrophobic alpha-helix that might function as a membrane anchor . Although hdrB and hdrC are juxtaposed, these genes are not near hdrA.

Eur J Biochem, 1994 Oct 1, 225(1), 235 - 42
Analysis of the phasing of four spectrin-like repeats in alpha-actinin; Gilmore AP et al.; Selected fragments of the central rod of chicken gizzard alpha-actinin were expressed as fusion proteins in Escherichia coli, with the aim of determining the positions in the sequence of the four successive spectrin-like repeats that make up this domain . The criteria for an independently folding unit were resistance to proteolysis and the high alpha helicity characteristic of the native protein . Sequences containing repeats 1-4, 2-4, 3-4 and 4 all generated stable fragments on digestion with trypsin and/or thermolysin and N-terminal sequencing gave the most probable starting position of each repeat . The sequences of all four inferred repeats and the sequences of the entire rod, were separately expressed and were shown to assume a stable, protease-resistant fold in solution . The repeat boundaries established in this way differed from those originally deduced from sequence alignments; the N-terminal boundaries of the repeats were 14-24 residues nearer the C-terminus than predicted . The ability to express individual repeats should facilitate identification of the binding sites for the cytoplasmic domains of beta 1 integrins and intercellular cell adhesion molecule-1 which have been localised to the rod domain of alpha-actinin.

Eur J Biochem, 1994 Oct 1, 225(1), 167 - 72
ATP synthesis catalyzed by the ATP synthase of Escherichia coli reconstituted into liposomes; Fischer S et al.; The H(+)-translocating F0F1-ATPase from Escherichia coli (EF0F1) was purified and reconstituted into preformed reverse-phase liposomes prepared from egg yolk phosphatidylcholine/phosphatidic acid . The EF0F1 liposomes were energized by an acid/base transition (pHout = 8.3; pHin = 5.0) and a superimposed K+/valinomycin diffusion potential ({K+}out = 100 mM; {K+}in = 0.6 mM) yielding a maximum rate (turnover number) of ATP synthesis of 27 +/- 8 mol ATP . mol EF0F1(-1) . s-1), i.e . 27 +/- 8 s-1 . This reaction was inhibited by NH4Cl or by addition of the F0F1 inhibitor N,N'-dicyclohexylcarbodiimide . The rate of ATP synthesis measured as a function of the phosphate and ADP concentrations, can be described by Michaelis-Menten kinetics with a Km of 0.7 +/- 0.2 mM for phosphate ({ADP} = 200 microM) and a Km of 27 +/- 7 microM for ADP ({phosphate} = 5 mM), respectively.

Eur J Biochem, 1994 Oct 1, 225(1), 143 - 9
Purification and characterization of the native and the recombinant Leishmania mexicana glycosomal glyceraldehyde-3-phosphate dehydrogenase; Hannaert V et al.; The gene coding for the glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana has been cloned into vector pET3A and expressed as a soluble and active protein in Escherichia coli BL21(DE3) in which the endogenous gene has been inactivated by mutation . The recombinant enzyme was purified to near homogeneity by ammonium sulphate precipitation, followed by hydrophobic and cation-exchange chromatography . From a 1-L culture of E . coli cells, 25 mg purified protein was obtained with a specific activity of 125 units/mg . The recombinant protein restores the natural E . coli phenotype when expressed at low level . The enzyme has also been partially purified from glycosomes of cultured L . mexicana promastigotes . The recombinant and the native proteins show identical mobilities on SDS/PAGE, and have the same isoelectric point and similar pH-activity profiles . The kinetics of both enzymes are very similar, the most important aspect being their lower apparent affinity for the cofactor NAD when compared to all other homologous enzymes studied, with the exception of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei.

Yeast, 1994 Oct, 10(10), 1267 - 72
A family of vectors that facilitate transposon and insertional mutagenesis of cloned genes in yeast; Allen JB et al.; This report describes two sets of plasmid vectors that facilitate the identification of regions of complementation in cloned genomic inserts via transposon or insertional mutagenesis . The first set contains ARS-H4 CEN6, a yeast selectable nutritional marker (HIS3, TRP1 or URA3), and neo for selection in Escherichia coli . These plasmids lack the Tn3 transposition immunity region present in pBR322 derived vectors, and are permissive recipients for Tn3 transposon mutagenesis . The second family of plasmids described facilitate gene disruption procedures performed in vitro . These vectors carry disruption cassettes that contain different yeast selectable markers (HIS3, LEU2, TRP1 or URA3) adjacent to the Tn5 neo gene . These genes can be excised as a cassette on a common restriction fragment and introduced into any desired restriction site with selection for kanamycin resistance.

J Bioenerg Biomembr, 1994 Oct, 26(5), 563 - 71
Relationship between the F0F1-ATPase and the K(+)-transport system within the membrane of anaerobically grown Escherichia coli . N,N'-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K(+)-transport; Trchounian AA et al.; A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K+ or Rb+, but not by Na+, over the range of zero to 100 mM was shown in the isolated membranes of E . coli grown anaerobically in the presence of glucose . This effect was observed only in parent and in the trkG, but not in the trkA, trkE, or trkH mutants . The trkG or the trkH mutant with an unc deletion had a residual ATPase activity not sensitive to DCCD . A stimulation of the DCCD-sensitive ATPase activity by K+ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate . Growth of the trkG, but not of other trk mutants, in the medium with moderate K+ activity did not depend on K+ concentration . Under upshock, K+ accumulation was essentially higher in the trkG mutant than in the other trk mutant . The K(+)-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grown E . coli has been shown to depend absolutely on both the F0F1 and the Trk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H(+)-K+ pump . The trkG gene is most probably not functional in anaerobically grown bacteria.

Eur J Cell Biol, 1994 Oct, 65(1), 220 - 8
Immunocytochemical localization of heterologously expressed adrenodoxin and its electron acceptor cytochrome P45011B1 in Escherichia coli; Erdmann B et al.; Adrenal steroid hydroxylase P45011B1 and its electron donor adrenodoxin were localized in the cortex of bovine adrenals by immunogold-silver staining . In order to test recently developed heterologous expression systems for both enzymes to enable structure-function studies, immunocytochemical marker methods were applied . Adrenodoxin, the ferredoxin of the adrenal gland, was successfully expressed and for the first time localized in Escherichia coli . By use of ultrathin cryosections and the protein A-gold technique, adrenodoxin was detectable in large amounts in the cytoplasm of the bacterial cells, and, following the insertion of the outer membrane protein A leader sequence of E . coli, also in the periplasmic space . A fusion protein between mature adrenodoxin and human P45011B1 was constructed and clearly localized in E . coli by antibodies against both proteins.

Eur J Cell Biol, 1994 Oct, 65(1), 200 - 5
Intracellular calcium alterations and free radical formation evaluated by flow cytometry in endotoxin-treated rat liver Kupffer and endothelial cells; Portoles MT et al.; During endotoxic shock, the liver exerts an endotoxin (lipopolysaccharide, LPS) clearance function with the participation of both sinusoidal (mainly Kupffer and endothelial cells) and parenchymal cells . In order to determine the specificity and diversity of response of each liver cell type, the effect of Escherichia coli 0111:B4 endotoxin (LPS) on intracellular Ca2+ content and reactive oxygen metabolite production in rat liver Kupffer, endothelial and parenchymal cells, was evaluated by flow cytometry during short treatment times (from 0-2 min) with a low dose of LPS (10 micrograms/ml) . Concerning sinusoidal cells, LPS produced a significant increase of intracellular Ca2+ in both endothelial and Kupffer cells . The LPS effect on Kupffer cells was higher than on endothelial cells . When intracellular reactive oxygen metabolite production was evaluated in LPS-treated sinusoidal cells, a fast and significant increase of Kupffer cells in activated state (cells with a high reactive oxygen intermediate production) was observed . However, endothelial cells did not show LPS-induced changes in their intracellular reactive oxygen metabolite content . All these results support a rapid activation of liver Kupffer cells by endotoxin consistent with the major role of this cellular type as active first line of defense during endotoxic shock . The liver endothelial cells are also involved in the first steps of the cell damage showing intracellular Ca2+ alterations . Liver parenchymal cells did not show any response at these experimental conditions (short treatment time and low LPS dose) indicating that longer treatment times are needed for LPS binding and action in agreement with previous studies.

Environ Health Perspect, 1994 Oct, 102 Suppl 6, 191 - 4
The carcinogenicity of methoxyl derivatives of 4-aminoazobenzene: correlation between DNA adducts and genotoxicity; Kojima M et al.; To elucidate the cause of the difference in genotoxic activity between carcinogenic 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and noncarcinogenic 2-methoxy-4-aminoazobenzene (2-MeO-AAB), we analyzed DNA adducts in the livers of rats exposed to either of these chemicals and studied the resulting biologic potential with the aid of in vitro modified M13 phage DNA . 32P-Postalbeling analysis revealed that the carcinogen 3-MeO-AAB produced 20-fold higher amounts of adducts than did 2-MeO-AAB . Five adducts were formed in the 3-MeO-AAB case whereas only one adduct was apparent in 2-MeO-AAB-treated rat . Studies of in vitro DNA replication using N-hydroxy (N-OH)-aminoazo dye-modified M13 phage DNA as a template demonstrated inhibition by 3-MeO-AAB adducts to be substantially greater than in the 2-MeO-AAB-adducts . The specificity of mutagenesis induced in M13mp9 phage DNA by these chemicals also was analyzed after transfection into SOS-induced Escherichia coli JM103, mutation frequencies being higher with N-OH-3-MeO-AAB- than N-OH-2-MeO-AAB-modified DNA . The mutation spectra differed in each case . Our data suggest that the difference in hepatocarcinogenic activity between the two chemicals depends not only on qualitative and quantitative variation in adduct formation but also on conformation changes in modified DNA.

J Biochem (Tokyo), 1994 Oct, 116(4), 923 - 30
Purification and characterization of Ca2+/calmodulin-dependent protein kinase IV kinase from rat brain; Okuno S et al.; Calmodulin-dependent protein kinase IV (CaM-kinase IV) kinase was recently discovered in the rat brain by its activity to activate the inactive recombinant CaM-kinase IV expressed in Escherichia coli {Okuno, S . and Fujisawa, H . (1993) J . Biochem . 114, 167-170} . In the present study, CaM-kinase IV kinase was purified approximately 2,000-fold from rat cerebral cortex by purification procedures including calmodulin affinity chromatography, and its properties were examined . The highly purified CaM-kinase IV kinase gave one major protein band corresponding to a molecular weight of about 66,000 upon SDS-PAGE . The purified CaM-kinase IV kinase phosphorylated and concomitantly activated CaM-kinase IV purified from rat brain as well as the recombinant kinase expressed in Escherichia coli in a Ca2+/calmodulin-dependent manner . The phosphorylation of CaM-kinase IV by CaM-kinase IV kinase occurred on only serine residue(s) . Among a number of proteins, including several known to be phosphorylated by the various protein kinases tested, CaM-kinase IV was the best substrate for CaM-kinase IV kinase . Since syntide-2, a synthetic peptide known to be a good peptide substrate for calmodulin-dependent protein kinase II (CaM-kinase II), was a fairly good substrate for CaM-kinase IV kinase, some kinetic properties of CaM-kinase IV kinase were examined using syntide-2 as a substrate . The Km value for the peptide substrate in the presence of Ca2+/calmodulin was almost two orders of magnitude lower than that in its absence, although the Vmax value was almost the same in the presence and absence of Ca2+/calmodulin.

J Biochem (Tokyo), 1994 Oct, 116(4), 916 - 22
Identification and characterization of the ackA (acetate kinase A)-pta (phosphotransacetylase) operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli; Kakuda H et al.; The pta gene encoding phosphotransacetylase was cloned on a high copy plasmid with or without the ackA gene encoding acetate kinase in Escherichia coli . The acetate kinase and phosphotransacetylase were overproduced in cells harboring the plasmid possessing both genes . Nucleotide sequencing of the pta gene revealed that it is able to produce a polypeptide comprising 714 amino acid residues, which starts at 70 base pairs downstream from the stop codon of the ackA gene . The 77-kDa protein band of overproduced phosphotransacetylase was observed on SDS-polyacrylamide gel electrophoresis, of which the amino terminal sequence corresponds to that of the deduced polypeptide without the amino terminal methionine . Two transcripts of pta of different sizes were found in the cells . A 3,700 nucleotide transcript, which covers the ackA and pta genes, seemed to be produced by the first promoter in the operon and a 2,300 nucleotide transcript, which covers just pta, seemed to be produced by the second promoter . In a synthetic medium containing acetate as the sole carbon source, the growth of an ackA-pta double mutant was greatly impaired . Complementation analyses revealed that both the acetate kinase and phosphotransacetylase were required for the rapid growth in the acetate medium.

J Biochem (Tokyo), 1994 Oct, 116(4), 877 - 81
Human centromere protein C (CENP-C) is a DNA-binding protein which possesses a novel DNA-binding motif; Sugimoto K et al.; Mammalian centromere proteins (CENPs) can be divided into those that translocate from centromere to midzone in the progress of mitosis, and those that remain at the centromere throughout the cell cycle . The latter including CENP-A, CENP-B, and CENP-C is the candidate for DNA-binding protein . CENP-B has been shown previously to possess the specific DNA-binding activity to 17-base pair sequences dispersed on human centromeric alphoid repeats . In this study, we examined DNA-binding property of CENP-C that is localized to inner kinetochore plate of the metaphase chromosome . We independently isolated a full-length cDNA encoding human CENP-C and expressed it as the polypeptide tagged with histidine oligomer in Escherichia coli . After affinity purification with Ni(2+)-chelated resin, DNA-binding activity of the recombinant CENP-C renatured on the membrane was demonstrated by using human genomic DNA and an alphoid subfamily in South-Western-type blotting analysis . By constructing a series of truncated products, the DNA-binding domain was located at an internal 101-amino-acid stretch with no apparent homology to any other DNA-binding proteins . This may suggest that CENP-C is directly involved in formation of kinetochore chromatin fibers.

J Biochem (Tokyo), 1994 Oct, 116(4), 838 - 44
Site- and strand-specific nicking at oriT of plasmid R100 in a purified system: enhancement of the nicking activity of TraI (helicase I) with TraY and IHF; Inamoto S et al.; We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100 . Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, TraI and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the TraI protein and Mg2+ . The products were complex DNA molecules with a protein covalently linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation . The same complex DNA molecules were formed in the presence of the TraI protein alone, indicating that the protein attached at the 5' end of the nick is the TraI protein . Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the TraI and TraY proteins at oriT.

J Biochem (Tokyo), 1994 Oct, 116(4), 826 - 32
Role of transit peptide sequence of plastocyanin for its expression, processing, and copper-binding activity in Escherichia coli; Hibino T et al.; Plastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast . To characterize the transit peptide of plastocyanin and develop expression systems for it in Escherichia coli, three kinds of expression vectors which encode different size precursor plastocyanin molecules were constructed . Their expression, processing, and copper-binding activity have been examined . When the full-length cDNA encoding the precursor plastocyanin from Silene pratensis was expressed in E . coli, a large amount of precursor plastocyanin accumulated in insoluble aggregates . Its accumulation level was increased by the addition of copper ions . About six percent of precursor plastocyanin molecules were transported into the periplasmic space and processed to the mature protein . On the other hand, expression of the intermediate size cDNA, which contains the hydrophobic domain and basic amino acid of C-terminal transit peptide, caused exclusive translocation to the periplasmic space and correct processing to the mature size . The addition of copper ions increased the holo-protein content, but did not change the polypeptide content of mature plastocyanin, indicating that translocation and processing are independent of the incorporation of copper ions . The mature plastocyanin content corresponds to 8% (w/w) of the total E . coli protein content (123 mg per liter of culture) . The purified mature holo-protein showed almost the same spectroscopic and kinetic properties as those of purified spinach plastocyanin . Expression of the cDNA encoding the mature polypeptide and two preceeding amino acid residues caused the accumulation of only a small amount of plastocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)

J Anim Sci, 1994 Oct, 72(10), 2661 - 9
The effect of dietary protein on performance and immune response in weanling pigs subjected to an inflammatory challenge; van Heugten E et al.; A total of 96, 21-d-old, crossbred weanling pigs (average initial weight was 6.0 kg) were assigned to one of six treatments to investigate the effect of dietary protein on performance and immune function of Escherichia coli lipopolysaccharide (LPS)-challenged and unchallenged pigs . A control diet was formulated to contain 14.7 MJ of DE/kg, 14 g of CP/MJ of DE, and 7 g of lysine/100 g of CP . Diets low and high in protein were formulated by changing protein levels to 60 or 120% of the control diet . On d 7 and 21, pigs were challenged with either a LPS solution or a saline solution . Lymphocyte blastogenesis was measured 2 d after LPS challenges and antibody response to sheep red blood cells (SRBC) or ovalbumin was measured 3 d after the challenges . Gain and feed consumption were determined 3 d after each LPS injection and at weekly intervals for a total period of 5 wk . Injection of LPS decreased daily gain, feed intake, feed efficiency, and efficiency of protein utilization (P < .05) . No interactive effects between LPS challenge and dietary protein were observed for pig performance (P > .10) . Daily gain and feed efficiency were improved when protein level was increased from 60 to 100% of the control diet (P < .01) . Efficiency of protein utilization for weight gain was lower when the 120% protein diet was fed (P < .01) . Antibody response to SRBC or ovalbumin was not affected by treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Genet, 1994 Oct, 26(4), 352 - 8
Cointegration of transforming DNAs in Aspergillus nidulans: a model using autonomously-replicating plasmids; Aleksenko AY; Transforming DNAs form cointegrates in Aspergillus nidulans by homologous and non-homologous recombination as well as by end-to-end ligation of linear fragments . This process has been studied by means of a model in which the linkage of a marker gene to the origin of autonomous replication AMA1 was selected for . Recombinant plasmids were rescued into Escherichia coli and subjected to restriction mapping and sequence analysis . It was shown that circular DNA molecules recombined predominantly within homologous fragments . Linear DNA fragments integrated into circular plasmids by invasion of their ends into random non-homologous sites, but exhibited some bias in choice of a target sequence . Cointegrates of multiple plasmid copies were often observed . In some of the plasmids analysed, short duplications of the target sequence flanking an inserted linear DNA fragment have been revealed.

Mol Mar Biol Biotechnol, 1994 Oct, 3(5), 235 - 42
Foreign gene transfer into nigorobuna (Carassius auratus grandoculis); Ueno K et al.; Supercoiled and linear plasmid DNA containing the Escherichia coli beta-galactosidase gene was injected into dechorionated nigorobuna (Carassius auratus grandoculis) eggs prior to first cleavage . The survival rates in the hatching stage were 73% to 89% for injected eggs in comparison with controls (non-injected chorionated eggs) . The exogenous DNA was detected by polymerase chain reaction (PCR) in all of the three-day-old larvae analyzed and in about 20% of two-year-old adult fish . Expression of the transgene was easily examined by a histochemical method using dechorionated eggs . The incidence of beta-galactosidase-positive embryos was highest in the gastrula or embryonic-body-formation stage and became low in the embryonic-body-movement stage . These results suggest the usefulness of funa (Carassius fishes) as a model fish in transgenic experiments, and the applicability of the transgenic technique to the improvement of funa as a food source.

Zhongguo Zhong Yao Za Zhi, 1994 Oct, 19(10), 626 - 8, 640
{Experimental study on compatible application of heat-clearing and detoxifying drugs with blood circulation improving drugs}; Ren Y et al.; The article describes the effectiveness of compatible application of heat-clearing and detoxifying drugs with blood circulation improving drugs to the animal model with endotoxemia and nonspecific inflammation . The compatible application reduces PGE2, endotoxin blood concentration and reduced viscosity of whole blood, decreases Evans blue extravasation volume and pes swelling percentage, increases serum cortisol content and enhances fibrinolytic activity . The experimental result shows that in most cases these two drugs work better when used in combination which implies that compatible application is more effective in detoxification, antiinflammation and inflammation recovery.

Neuroscience, 1994 Oct, 62(3), 721 - 39
Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine1A receptor; Gerard C et al.; The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum . The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine1A receptor antibodies . Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection . These antibodies only marginally affected the specific binding of {3H}8-hydroxy-2-(di-n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine1A receptors in rat hippocampal membranes . However, antibodies were able to immunoprecipitate 5-hydroxytryptamine1A receptor binding sites solubilized from rat hippocampal membranes . The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei . The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine1A receptors might differ from one neuronal cell type to another.

Mol Biochem Parasitol, 1994 Oct, 67(2), 281 - 8
Induction of the iron-containing superoxide dismutase in Entamoeba histolytica by a superoxide anion-generating system or by iron chelation; Bruchhaus I et al.; The regulation of superoxide dismutase (SOD) expression was studied in 4 Entamoeba histolytica isolates . In comparison to anaerobic conditions, cultivation of the amoebae in the presence of superoxide radical anions or a ferrous iron chelator revealed substantial increase of SOD expression . Under the different culture conditions, all SOD activity could be exclusively attributed to an iron-containing type (FeSOD) . Northern blot analysis revealed that FeSOD expression was regulated on the transcriptional level . Within the 5'-flanking region of the amoebic FeSOD gene, a 19-bp fragment was found with 68% sequence identity to the consensus motif of the binding site for the ferric uptake regulation gene product of Escherichia coli . Electrophoretic mobility shift assays with this 19-bp fragment and with amoebic nuclear extracts revealed specific DNA/protein complex formation . The results indicate that the regulation of E . histolytica FeSOD expression is similar to that of the manganese-containing SOD (MnSOD) of E . coli.

J Virol Methods, 1994 Oct, 49(3), 285 - 94
Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus; Carn VM et al.; The gene coding for the capripoxvirus structural protein P32 was cloned, expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and purified on glutathione Sepharose . An indirect enzyme linked immunosorbent assay (ELISA) using this antigen was developed to screen bovine sera for antibodies to capripoxvirus . Sequential serum samples from experimentally infected animals tested by ELISA and by virus neutralisation test (VNT) showed that the ELISA was more sensitive and detected antibodies to capripoxvirus earlier post-infection than the VNT.

J Virol Methods, 1994 Oct, 49(3), 257 - 68
Production of measles nucleoprotein in different expression systems and its use as a diagnostic reagent; Warnes A et al.; Measles nucleoprotein has been successfully expressed in three different hosts, bacterial (Escherichia coli BL21 DE3), insect (Spodoptera frugiperda; Sf9) and mammalian (primary human fibroblasts) cells, each producing an antigenic protein of M(r) 60 kDa . The nucleoprotein produced in all three hosts was used in an ELISA for the detection of antibodies to measles virus in a cohort of haemagglutinin-positive or -negative human sera . Data produced from baculovirus and adenovirus-based antigens indicated that there was good correlation between the ELISA results and previous haemagglutination inhibition test data, and there was little background interference by cellular proteins or the development of false positive or negative results . The assay was rapid as it could be carried out in under 4 h, sensitive as the sera could be diluted by at least 1000-fold, and versatile as both IgG and IgM could be detected and differentiated.

Eur J Clin Chem Clin Biochem, 1994 Oct, 32(10), 797 - 803
Assay of endotoxin in human plasma using immobilized histidine, Limulus amoebocyte lysate and chromogenic substrate; Minobe S et al.; The Limulus amoebocyte lysate test for endotoxin is inhibited or enhanced by many substances . It is particularly difficult to determine endotoxin in plasma . In order to overcome this problem, we have modified the specific endotoxin assay method by using a membrane filter unit, a chromogenic Limulus amoebocyte lysate reagent, and immobilized histidine (which is a specific adsorbent for endotoxins) . This immobilized histidine method consists of the endotoxin adsorption step on immobilized histidine, the separation step, in which Limulus amoebocyte lysate-interfering substances are removed, and the Limulus amoebocyte lysate test . Preheating of plasma samples (40-fold dilution with distilled water, at 100 degrees C for 7.5 min) was necessary, and it was necessary to dilute the sample more than 100-fold for the adsorption step . Under these conditions, the fraction of endotoxin recovered from plasma by the immobilized histidine method was almost 1 . Moreover, by increasing the sample volume and extending the Limulus amoebocyte lysate reaction time, the sensitivity could be increased . By using the immobilized histidine method, 50-200 units/l of endotoxin in plasma samples can be accurately assayed . The method was used for the determination of plasma endotoxin in rabbits.

New Microbiol, 1994 Oct, 17(4), 291 - 6
Molecular cloning of Chlamydia trachomatis 26K protein expressed in Escherichia coli; Del Pezzo M et al.; Molecular genetics appears to be the most promising approach to understanding the biology and pathology of Chlamydia . This report focuses on the cloning and the protein expression of a DNA fragment from Chlamydia trachomatis DK20 chromosome . Results of hybridization experiments suggest that this sequence is specifically present within chlamydial DNA . The coding capacity of this DNA fragment is supported by the expression of a 26,000 m.w . peptide, in an Escherichia coli maxicell system.

Hybridoma, 1994 Oct, 13(5), 373 - 81
Monoclonal antibody specifically reacting against 73-kilodalton heat shock cognate protein: possible expression on mammalian cell surface; Tsuboi N et al.; The heat shock proteins (hsp) are regarded as being immunogenic to the animal hosts . Although certain hsp are suggested to be expressed on the cell surface, further evidence for the cell surface expression of these proteins has been required . In this article we report the development of a MAb NT22 . This antibody reacted with ATP-binding proteins (which contain a large amount of 70-kDa hsp family) of HeLa cells, and with purified bovine 70-kDa hsp . It did not react with the E . coli lysate, but clearly reacted with the recombinant rat hsc73 . However, NT22 failed to react with hsp72 . Furthermore, stress treatment of cells also indicated that considerable amounts of NT22-defined antigen translocated into the nucleus from the cell cytoplasm . These results suggest that NT22 is a novel MAb that reacts specifically to the mammalian hsc73 . Moreover, this antibody could detect the constitutive and stress-induced cell surface expression of its relevant antigen . It is expressed preferentially on EBV-transformed B cell and certain epithelial cancer cell lines . However, resting B cells did not express this antigen on the cell surface . These data indicate that hsc73 could be expressed on the cell surface of certain cells, and suggest that hsc73 may interact with the host immune system.

Lymphokine Cytokine Res, 1994 Oct, 13(5), 271 - 5
Hyperthermia induces IL-1 alpha but does not decrease release of IL-1 alpha or TNF-alpha after endotoxin; Blake D et al.; Heat treatments administered prior to the onset of sepsis or endotoxemia markedly increase survival . A potential mechanism for the beneficial effect of heat could be effects on IL-1 alpha and TNF-alpha, important mediators of sepsis and endotoxemia . Administration of IL-1 or TNF prior to development of sepsis and endotoxemia increases survival; thus, prophylactic heat treatments may protect by releasing IL-1 or TNF . Paradoxically, an alternative mechanism of protection of prophylactic heat treatments could be to decrease the amount of IL-1 and TNF released during sepsis or endotoxemia . Cells pretreated with heat do not produce as much IL-1 or TNF in response to endotoxin as cells that have not been pretreated with heat . The purpose of this investigation was to determine if hyperthermia caused release of cytokines and/or blunted the rise in cytokines occurring after endotoxin . Mice were anesthetized with ketamine/xylazine and immersed in a water bath at 37.0 or 42.0 degrees C for sham or heat treatments . At 6-7 h after recovery from anesthesia and immersion, sham and heat-treated mice were injected with Escherichia coli endotoxin . Both heat-treated and sham mice had elevated plasma IL-1 alpha 2 h after anesthesia and immersion but IL-1 alpha was approximately 3-fold greater in the heated mice, 732 +/- 50 vs . 256 +/- 76 pg/ml (p < 0.01) . Blood samples obtained after endotoxin revealed no difference in levels of TNF-alpha (5477 +/- 742 vs . 6514 +/- 652 pg/ml) or IL-1 alpha (546 +/- 72 vs . 603 +/- 121 pg/ml) in the sham vs . heated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1994 Oct, 7(10), 1267 - 76
An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions; Taylor MA et al.; The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time . This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli . Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained . Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate . At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants . Only two pKa values, however, are detectable in the acid limb using the Ala mutant . Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence . Changing the position of this ionizable group in any way reduces activity . Complete removal of the charged group reduces catalytic competence even further . This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

J Vet Pharmacol Ther, 1994 Oct, 17(5), 369 - 73
Pharmacokinetics of gentamicin following single dose intravenous administration in normal and febrile goats; Ahmad AH et al.; A pharmacokinetic study of gentamicin (5 mg/kg intravenous (i.v.)) was conducted first in clinically healthy female goats and then in the same goats after induction of fever by Escherichia coli endotoxin (0.2 microgram/kg i.v.) . Rectal temperature increased 1 degrees to 1.5 degrees C in febrile goats . Differences in the blood serum concentrations of gentamicin were not observed at any time between febrile and normal goats . The disposition kinetics of gentamicin were described by a biexponential expression CP = Ae-alpha t + Be-beta t . Median values for the half-lives of gentamicin were 103.6 min in normal and 136.0 min in febrile goats . The apparent volume of distribution (Vd) was 263.3 ml/kg in the febrile goats which was not different from that in the normal goats (240.6 ml/kg) . The volume of the central compartment (Vc) was almost identical in normal and febrile goats . The body clearance (Cl beta) was observed to be 1.7 and 1.6 ml/min.kg in normal and febrile goats, respectively . Dosage regimens for gentamicin were calculated on the basis of median kinetic data.

J Biochem Toxicol, 1994 Oct, 9(5), 225 - 34
Characterization of heterologously expressed recombinant retinoic acid receptors with natural or synthetic retinoids; Kim YW et al.; The first step in retinoid action is binding to their nuclear receptors . Therefore, characterization of binding characteristics of retinoids is of major importance . Human retinoic acid receptors alpha (hRAR alpha), hRAR beta, and mouse RAR gamma (mRAR gamma) were expressed heterologously in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein . The expressed fusion proteins were functional and bound specifically to the all-trans-retinoic acid (RA) . The dissociation constants (Kd) for RA were 1.4 nM for GST-hRAR alpha, 1.4 nM for GST-hRAR beta, and 3.3 nM for GST-mRAR gamma, respectively . The fusion proteins were further used for competitive displacement assays to determine the displacement constant (DC50) for other selected retinoids . All-trans-RA and 4-oxo-all-trans-RA have high affinity with all three receptors (DC50 = 0.8-55 nM) . The 13-cis RA binds to hRAR alpha with low affinity, but not to other RARs evaluated here . All-trans-N-ethylretinamide, all-trans-retinylacetate, and an ethyl ester of tetrahydronaphthalene derivative had no affinity to any RARs . The hRAR alpha and mRAR gamma receptors did not bind a naphthalene carboxylic acid derivative of RA, but hRAR beta binds this chemical with high affinity . Results indicated that the three recombinant proteins were functional in binding various RA congeners . The affinity and binding data of these retinoids were compared to their observed teratogenic activity.

Hepatogastroenterology, 1994 Oct, 41(5), 432 - 7
Hepatic mitochondrial changes in experimental obstructive jaundice complicated by biliary infection; Nagano I et al.; The effects of biliary infection on the structural and functional changes in the liver in obstructive jaundice was studied using rats as experimental animals . Biliary infection with obstructive jaundice was induced in the animals by injecting Escherichia coli into the common bile duct through a nylon tube cannulated into the duct . This was followed by the clamping of the tube . For the controls, the tube was clamped in the absence of Escherichia coli . After 3, 7, 14 and 21 days, the animals were sacrificed, and some serum enzyme activities, histological changes in the liver, and the coupling efficiency of mitochondria isolated from the liver were investigated . The phosphorylating ability of hepatic mitochondria was more seriously affected when the obstruction was complicated by cholangitis . We suggest that, when associated with obstructive jaundice, biliary infection should be carefully treated prior to hepatectomy.

Curr Biol, 1994 Oct 1, 4(10), 945 - 6
Replication initiation . A new controller in Escherichia coli; Baker TA; A new factor involved in the sequestration of recently active replication origins in Escherichia coli, SeqA, has been discovered and appears to be a multifaceted negative regulator of replication initiation.

Curr Biol, 1994 Oct 1, 4(10), 884 - 91
Three-dimensional solution structure of the pleckstrin homology domain from dynamin; Downing AK et al.; BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown . The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems . Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them . RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP . The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees . The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99) . A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli . CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains . This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold . On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.

Hum Mol Genet, 1994 Oct, 3(10), 1807 - 10
Characterization and expression of cDNA encoding coproporphyrinogen oxidase from a patient with hereditary coproporphyria; Fujita H et al.; Hereditary coproporphyria (HCP) is an acute hepatic porphyria with autosomal dominant inheritance, but with a variable degree of clinical expression . Molecular cloning, sequencing and expression of the defective gene for coproporphyrinogen oxidase (CPO) in a patient with HCP were carried out . Enzyme assays revealed that CPO activity in EBV-transformed lymphoblastoid cells from the proband and one of her sisters was approximately 50% of normal . Nucleotide sequence analysis of CPO cDNAs isolated from the proband's cells demonstrated three base substitutions, and three accompanying amino acid substitutions . An A514-->C transition causing a Asn172-->His substitution occurred in one allele, while two other transitions, G265-->A and G580-->A, caused Gly89-->Ser and Val194-->Ile substitutions, respectively, in the other allele . The A514-->C and the G580-->A transitions are known genetic polymorphisms . Transfection of CPO cDNA into Escherichia coli demonstrated that cDNA with the G265-->A transition produced a protein with less than 5% of normal enzyme activity . These findings indicate that the G265-->A transition, involving the highly conserved glycine residue at the 89th position, is responsible for the CPO defect in the patient and accounts for the partial deficiency of CPO activity in this pedigree.

Protein Sci, 1994 Oct, 3(10), 1746 - 59
Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis; Witmer MR et al.; The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis . The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations . Unadenylylated and fully adenylylated enzyme forms were studied . The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H . For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D) . Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold) . Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS . We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate . Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release . Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps . Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion . The data are discussed in the context of the known X-ray structures of GS.

AIDS Res Hum Retroviruses, 1994 Oct, 10(10), 1231 - 40
Fusogenic activity of amino-terminal region of HIV type 1 Nef protein; Curtain CC et al.; We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E . coli . Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated . Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching . Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures . Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra . On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum . Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer . Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets . The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein . For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles . The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

Nat Genet, 1994 Oct, 8(2), 122 - 8
Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA; Suzuki Y et al.; Holocarboxylase synthetase (HCS) plays an essential role in biotin utilization in eukaryotic cells and its deficiency causes biotin-responsive multiple carboxylase deficiency in humans . We have cloned the human HCS cDNA and show that antiserum against the recombinant protein immunoprecipitates human HCS . A one base deletion resulting in a premature termination and a missense mutation (Leu to Pro) were found in cells from siblings with HCS deficiency . Human HCS shows homology to BirA, which acts as both a biotin-{acetyl-CoA-carboxylase} ligase and a biotin repressor in E . coli, suggesting a functional relationship between the two proteins . The human HCS gene maps to chromosome 21q22.1.

J Rheumatol, 1994 Oct, 21(10), 1951 - 4
Phospholipase A2 in juvenile rheumatoid arthritis: correlation to disease type and activity; Pruzanski W et al.; OBJECTIVE . Secretory nonpancreatic phospholipase A2 (snpPLA2) is a proinflammatory enzyme and its activity in serum correlates with disease activity in adults with rheumatoid arthritis . Juvenile rheumatoid arthritis (JRA) may be stratified into 3 clinical types with differing degrees of disease activity . Since in JRA there are no reliable indices of disease activity, our objective was to find whether the level of circulating snpPLA2 correlates with the severity of inflammation and with JRA activity . METHODS . PLA2 enzymatic activity was assayed using E . coli membranes labelled with (14C)-oleic acid . SnpPLA2 immunoreactivity was tested by ELISA technique using monoclonal antibodies against recombinant human (rh) snpPLA2 . SnpPLA2 activity was determined in sera of 127 children including 25 with systemic (S-JRA), 50 with polyarticular (Po-JRA) and 52 with pauciarticular (Pa-JRA) types of JRA . Twenty-five patients with active disease, were subsequently restudied in an inactive phase . RESULTS . Markedly increased snpPLA2 (> mean + 2 SD of normal mean, i.e., > 575 U/ml) was found during the active disease in 100% S-JRA, 57% Po-JRA and 25% Pa-JRA patients . The differences in the mean and median PLA2 activity among these 3 subtypes of JRA were highly significant (p < 0.001) with the highest levels found in S-JRA and the lowest in Pa-JRA . Presence of rheumatoid factor and/or of antinuclear antibody had no relation to the level of snpPLA2 . SnpPLA2 activity became markedly lower when active inflammation became quiescent . In the whole group, snpPLA2 activity correlated highly with the Lansbury index, number of involved joints and number of effusions . A significant positive correlation was also found between snpPLA2 and erythrocyte sedimentation rate (ESR) and neutrophil count, while a significant negative correlation was noted with the level of albumin and hemoglobin . With the exception of snpPLA2, other laboratory variables did not correlate with the number of effusions or the number of active joints . However a negative correlation was noted between both hemoglobin and albumin, and Lansbury index . CONCLUSION . Circulating snpPLA2 significantly correlates with JRA activity and may serve as an index of activity in JRA especially in patients with systemic type of disease.

J Dairy Sci, 1994 Oct, 77(10), 3124 - 31
Effects of housing and colostrum feeding on the prevalence of selected infectious organisms in feces of Jersey calves; Quigley JD 3rd et al.; Neonatal Jersey calves (n = 96) were used to evaluate effects of housing (individual hutches or wooden pens in a barn) and colostrum feeding (calves were separated from the dam and fed 2 L of colostrum in nipple-bottles or allowed to nurse the dam for 3 d) on the prevalence of selected organisms in feces . Prevalence of Cryptosporidium and Eimeria were reduced, and prevalence of rotavirus tended to be reduced, when calves were housed in hutches . Prevalence of coronavirus was unaffected by treatment . Weekly prevalence of Giardia was increased when calves were left to nurse the dam for 3 d . Mean prevalence of Cryptosporidia (wk 1 to 4), Eimeria (wk 4 to 5), Giardia, rotavirus, and coronavirus (wk 1 to 5) were 34.7, 20.6, 27.1, 15.8, and 4.9%, respectively . Escherichia coli (K99 positive) were observed in 3 of 174 samples cultured . Methods of housing and colostrum feeding affected acquisition of enteropathogens in this study.

Virus Res, 1994 Oct, 34(1), 49 - 61
Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence; Kochneva GV et al.; Three independently selected spontaneous thymidine kinase-negative mutants (TK-phenotype) and a recombinant with Escherichia coli beta-galactosidase gene (LacZ+ phenotype) inserted in the viral thymidine kinase gene (tk) were derived from a plaque-cloned isolate of K-1 ectromelia virus strain (TK+ phenotype) . Dramatically decreased virulence of TK- variants was observed for all routes of mouse inoculation . The kinetics of TK+ and TK- variants in various target organs indicated a significant decrease of production and dissemination of TK- mutants and recombinant in the organs of mice . In the spleen and liver of intranasally or intracerebrally infected mice TK- virus was not detected during the entire period of observation . Analysis of organs homogenates of mice intranasally infected by a mixture of recombinant with TK-LacZ+ phenotype and parental isolate with TK+LacZ- phenotype on the monolayers of TK- cells indicated that only white plaques (LacZ-) with the TK+ phenotype appeared from liver and spleen homogenates . Thus, the mouse acts as a live filter much more efficiently than any other selective systems . Ultrastructural studies showed that viral damage in animals infected by TK- variants was far less than that observed in mice, infected with wild type of ectromelia virus and pathological lessions were slight and reversible . Replication of ectromelia virus TK- variants was blocked at the viroplasma stage in cells with a high level of differentiation in contrast to TK+ variants . Most likely, such restriction of target cells assortment is the general reason of reduced virulence in the case of tk-gene inactivation.

Mol Microbiol, 1994 Oct, 14(2), 335 - 46
Multiple protein-DNA and protein-protein interactions are involved in transcriptional activation by MalT; Danot O et al.; The promoters of the Escherichia coli maltose regulon are positively regulated by the MalT protein, which recognizes a short asymmetric nucleotide sequence that is present as several copies in each promoter of the regulon . We report a detailed biochemical characterization of the interaction of MalT with the promoter of the malPQ operon . The MalT sites in malPp were precisely located and their occupation as a function of MalT concentration was quantified using DNase I and dimethyl sulphate footprinting experiments . The contribution of each site to malPp activity was assessed by introducing mutations that destroy them and measuring the residual in vivo and in vitro activity . Two main results were obtained . First, although the proximal MalT site is centred at -37.5, RNA polymerase is likely to establish a contact required for malPp activity with at least one base pair of the promoter -35 region; this close proximity between RNA polymerase and MalT bound to site 1 suggests that the two proteins interact . Second, even if the interaction of MalT with the three functional sites in malPp is a co-operative process, the MalT molecules bound to the two distal sites play a more subtle role than simply increasing the occupancy of the proximal site and may also contact RNA polymerase . We suggest that, in the nucleoprotein structure responsible for the initiation of transcription, MalT, RNA polymerase and malPp are held together by several weak interactions.

Mol Microbiol, 1994 Oct, 14(2), 309 - 21
The tail of a chaperonin: the C-terminal region of Escherichia coli GroEL protein; McLennan NF et al.; The active form of the HSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven-membered rings . We have used site-directed mutagenesis to construct forms of the 547-amino-acid monomer truncated at the C-terminus . We show here that forms that are 520 amino acids long or longer are close to being fully functional . Removing one further amino acid, however, results in a protein, GroEL519, which retains little function . This truncated form is metabolically stable but is not recovered from the cell in particle form . When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell . When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly-competent forms of GroEL . This can be seen as partial complementation of the temperature-sensitive mutant groEL44 . We conclude that amino acid 520 is crucial for particle assembly . GroEL516 has in vivo properties similar to those of GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.

Mol Microbiol, 1994 Oct, 14(2), 263 - 70
Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli; Ravn P et al.; We have studied the formation of spontaneous mutations on plasmids present in the monomeric and dimeric states in a recF strain of Escherichia coli . Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322-derived plasmid pPY97 . The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence . No apparent mutational hot-spot was found . The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids . Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322 . A mechanisms to account for this is suggested . Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid . Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.

Mol Microbiol, 1994 Oct, 14(1), 7 - 16
Altered pH and lysine signalling mutants of cadC, a gene encoding a membrane-bound transcriptional activator of the Escherichia coli cadBA operon; Dell CL et al.; The Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine . cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion . Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA-binding domain in the amino terminus from a periplasmic domain . Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH 5.8) or at a non-inducing pH (pH 7.6) . Eight cadC mutants were isolated based on their ability to confer expression of a cadA-lacZ fusion independent of low external pH or exogenous lysine . Five of these mutants expressed the cadA-lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence at pH 5.8 but not at pH 7.6 . These results support a model in which CadC is a membrane-bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.

Mol Microbiol, 1994 Oct, 14(1), 51 - 60
Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis; Pizza M et al.; Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity . Following site-directed mutagenesis, the mutants obtained could be divided into three groups . The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu . This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity . The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr . The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn . The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.

Mol Microbiol, 1994 Oct, 14(1), 31 - 40
Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli; Benard L et al.; Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site . By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding . The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot . It is deduced that a specific pseudoknot conformation is a key element for autoregulation.

Mol Microbiol, 1994 Oct, 14(1), 151 - 61
Escherichia coli tyrT gene transcription is sensitive to DNA supercoiling in its native chromosomal context: effect of DNA topoisomerase IV overexpression on tyrT promoter function; Free A et al.; We have investigated the in vivo DNA supercoiling sensitivity of the Escherichia coli tRNA(1tyr) gene (tyrT) promoter in its normal chromosomal location . Here, the native tyrT promoter is found to be exquisitely sensitive to mutations and to drugs which alter the level of DNA supercoiling . We show that the response of the tyrT promoter to supercoiling is qualitatively similar to that of a known supercoiling-sensitive tRNA gene promoter, hisR . Specifically, treatments which increase in vivo DNA supercoiling levels enhance transcription of these tRNA genes . Particularly striking is the strong enhancement of expression from both promoters by a transposon insertion mutation in the topA gene encoding DNA toposisomerase I . This phenotypic effect can be complemented by providing active topoisomerase I in trans from a recombinant plasmid . Interestingly, it can also be complemented by overexpression of the genes encoding the subunits of DNA topoisomerase IV . We believe that this is the first demonstration that DNA topoisomerase IV can influence gene expression and it suggests that DNA topoisomerase I is partially redundant, at least in E . coli.

Genetics, 1994 Oct, 138(2), 263 - 70
Spontaneous mutation in Escherichia coli containing the dnaE911 DNA polymerase antimutator allele; Oller AR et al.; We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity . These mutants have a mutation in the dnaE gene, encoding the alpha subunit of DNA polymerase III . They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors . In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background . In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways . Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation . The data show that dnaE911 decreases the level of Rifr, lacI and galK mutations in this background by 1.5-2-fold . DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity . Transversions are decreased by approximately 3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged . Among the transversions, A.T-->T.A are affected most strongly (approximately 6-fold) . In addition to this effect on transversions within the lacI gene, one previously recognized A.T-->G.C base-pair substitution hotspot in the lac operator is also reduced (approximately 5-fold) . The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.

Genetics, 1994 Oct, 138(2), 253 - 61
Population dynamics of a Lac- strain of Escherichia coli during selection for lactose utilization; Foster PL; During selection for lactose utilization, Lac+ revertants of FC40, a Lac- strain of Escherichia coli, appear at a high rate . Yet, no Lac+ revertants appear in the absence of lactose, or in its presence if the cells have another, unfulfilled requirement for growth . This study investigates more fully the population dynamics of FC40 when incubated in the absence of a carbon source or when undergoing selection for lactose utilization . In the absence of a carbon source, the viable cell numbers do not change over 6 days . When incubated in liquid lactose medium, Lac- cells do not undergo any measurable increase in numbers or in turbidity for at least 2 days . When FC40 is plated on lactose minimum medium in the presence of scavenger cells, the upper limit to the amount of growth of Lac- cells during 5 days is one doubling, and there is no evidence for turnover (i.e., a balance between growth and death) . The presence of a minority population that could form microcolonies was not detected . The implications of these results, plus the fact that the appearance of Lac+ revertants during lactose selection is nearly constant with time, are discussed in reference to several models that have been postulated to account for adaptive mutations.

Protein Expr Purif, 1994 Oct, 5(5), 509 - 17
In vivo biotinylated recombinant antibodies: construction, characterization, and application of a bifunctional Fab-BCCP fusion protein produced in Escherichia coli; Weiss E et al.; We describe a novel vector system suitable for the efficient preparation of in vivo biotinylated antibody Fab fragments in Escherichia coli . The previously described pGE20 vector used for the functional expression of truncated heavy (Fd) and light (L) chains of Fab into the bacterial culture medium was modified by inserting the C-terminal 101-amino-acid polypeptide of the biotin carboxyl carrier protein subunit of E . coli acetyl-CoA carboxylase (BCCP*) . The secreted Fd-BCCP* fusion and L chain proteins were found to be disulfide linked and Fab-BCCP* complexes of an IgG1 antibody (Mab4) to human tumor necrosis factor alpha (TNF) were shown to retain both antigen and streptavidin-binding activities . The capacity of the Fab4 linked to BCCP* to bind TNF was identical to that observed with unmodified Fab4 . Up to 15% of the expressed hybrids were able to interact with streptavidin when exogeneous d-biotin was added into the bacterial culture medium . The Fab4-BCCP* molecules were found to be more efficient than Fab4 linked to an engineered streptavidin-affinity tag for the detection of antigen on solid phase . In addition, we show here that the bacterially expressed Fab4-BCCP* complexes, adsorbed to streptavidin-agarose beads, can be used for the one-step purification of recombinant TNF by immunoaffinity chromatography.

Protein Expr Purif, 1994 Oct, 5(5), 468 - 75
Purification of recombinant human transcription factor IIB by immunoaffinity chromatography; Thompson NE et al.; The human RNA polymerase II transcription factor IIB (TFIIB) was purified from a bacterial expression system by immunoaffinity chromatography . Mouse monoclonal antibodies (MAbs) were prepared that react with TFIIB . A modified enzyme-linked immunosorbent assay was used to screen for MAbs that release the antigen in the presence of a low molecular weight polyhydroxylated compound and a nonchaotropic salt (polyol-responsive MAbs) . One polyol-responsive MAb (designated IIB8) was purified by chromatography on protein A and conjugated to cyanogen bromide-activated Sepharose 4B . Escherichia coli strain BL21 (DE3) containing the pLysS plasmid was transformed with the human TFIIB gene contained in the pET11a vector (phIIB) . After induction with IPTG, the cells were harvested and lysed . The lysate was treated with 0.5% polyethyleneimine and centrifuged . The supernatant fluid was applied to the IIB8-Sepharose . After extensive washing, the TFIIB was eluted with Tris-EDTA buffer containing 0.75 M ammonium sulfate and 40% propylene glycol . The purified TFIIB was active when added back to TFIIB-depleted HeLa nuclear extract and when used in the IgH minimal promoter system . This method will be useful for the rapid purification of TFIIB mutants and for the purification of large amounts of highly purified TFIIB for biochemical studies . In addition, this procedure establishes the general applicability of the use of polyol-responsive MAbs for the rapid, gentle purification of labile proteins.

Protein Expr Purif, 1994 Oct, 5(5), 458 - 67
Expression in Escherichia coli: production and purification of both subunits of the human general transcription factor TFIIE; Chalut C et al.; Both subunits of the human class II transcription factor TFIIE (rTFIIE alpha and rTFIIE beta) have been overexpressed in Escherichia coli at 26 degrees C using a T7 RNA polymerase expression system and further purified to apparent homogeneity . As in this system rTFIIE alpha was poorly expressed and copurified with a truncated form, we expressed rTFIIE alpha as a fusion protein . These overexpressed subunits of TFIIE are similar to the endogenous subunits according to the following criteria: molecular weight, microsequencing, and transcription activity.

Protein Expr Purif, 1994 Oct, 5(5), 442 - 8
Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization; Bukovska G et al.; Cystathionine beta-synthase (CBS) purification from mammalian tissues is complicated by proteolysis and enzyme aggregation . To surmount these difficulties, we cloned human CBS cDNA in tandem with the beta-galactosidase sequence of the fusion vector, pAX5-, then expressed the fusion protein, beta-galactosidase/CBS, in transformed Escherichia coli cells . Proteolytic treatment of the ammonium sulfate fraction of bacterial lysates with endoproteinase Xa liberated CBS which could then be separated from its fusion partner by DEAE-cellulose chromatography . This nearly homogeneous enzyme preparation was purified 140-fold over the crude bacterial lysate with nearly 50% recovery, and its specific activity, 210 U/mg protein, was comparable to that purified from human liver . The purified enzyme contained pyridoxal 5'-phosphate and exhibited positive cooperativity toward S-adenosyl-L-methionine (Hill coefficient = 5.2; Kact = 34 microM) . Km values of the cloned enzyme in the absence of AdoMet are 3.1 and 1.1 mM for serine and homocysteine, respectively . They are virtually identical to those from human hepatic CBS . A Soret absorbance band (lambda max = 428 nm) which shifted to 448 nm after reduction with sodium dithionite revealed the presence of heme in the enzyme . Expression of the fusion protein in E . coli with subsequent purification represents the first time this enzyme has been isolated in sufficient quantities for biophysical and biochemical investigation.

Protein Expr Purif, 1994 Oct, 5(5), 432 - 41
A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase; Fetzer J et al.; Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein . The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography . Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing . In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry . Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine . Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers . Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.

Ann Oncol, 1994 Oct, 5(8), 709 - 16
A dose intensity study of FLAC (5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide) chemotherapy and Escherichia coli-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) in advanced breast cancer patients; O'Shaughnessy JA et al.; BACKGROUND: It has been demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) can ameliorate chemotherapy-induced neutropenia . The extent to which GM-CSF can increase the actual delivered dose intensity of combination chemotherapy over multiple cycles of therapy to patients with advanced breast cancer has not been well defined . We conducted a phase I/II study of dose-intensive FLAC chemotherapy (5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide) in combination with GM-CSF in patients with locally advanced and metastatic breast cancer to study the acute and cumulative toxicities, anti-tumor activity and dose-intensity achievable with this regimen . METHODS: Eighty-one patients with newly diagnosed stages IIB, III and IV breast cancer who were previously untreated with chemotherapy and who had measurable disease received multiple cycles of FLAC chemotherapy plus E . coli-derived GM-CSF administered every three weeks . RESULTS: FLAC plus GM-CSF as associated with significant cumulative hematologic toxicity . Ninety-eight percent of patients developed grade 4 neutropenia; 29% of all cycles administered required hospitalization for fever and neutropenia; 41% and 22% of cycles required red blood cell and platelet transfusions, respectively . Other significant toxicities with E . coli-derived GM-CSF included mild to moderate first dose effects (hypotension, dyspnea, abdominal cramping) in 30% of patients; late occurring anaphylactoid reactions in 11% of patients; and vascular thromboses . The average delivered dose intensities over all cycles were cyclophosphamide, 210 mg/m2/week; doxorubicin, 14.8 mg/m2/week and 5-fluorouracil, 342 mg/m2/week . The overall clinical response rates were 100% and 83% for LABC and metastatic patients, respectively . There were 23% (6/26) pathologic CR's in the LABC patients given neoadjuvant FLAC and 22% (12/54) clinical CR's in the stage IV patients . The median survival of the LABC patients has not been reached (> 26 months) and is 30 months for the stage IV patients . CONCLUSIONS: The administration of multiple cycles of FLAC plus E . coli-derived GM-CSF therapy is associated with cumulative, dose-limiting myelosuppression, especially thrombocytopenia, as well as significant clinical toxicity . A modest increase in FLAC dose intensity over the starting doses was achievable with the addition of GM-CSF . FLAC chemotherapy has substantial antitumor activity in the treatment of advanced breast cancer . The potential usefulness of FLAC plus GM-CSF must be balanced by its considerable cost and alteration in patients' quality of life due to toxicity . Combination hematopoietic growth factor strategies may be able to reduce the toxicity of FLAC and to allow further increase dose intensity.

J Periodontol, 1994 Oct, 65(10), 937 - 41
Smokeless tobacco effects on monocyte secretion of PGE2 and IL-1 beta; Payne JB et al.; The use of smokeless tobacco (ST) products is associated with mucosal lesions, gingival recession, and attachment loss at the site of tobacco placement . Monocytes/macrophages are primary producers of PGE2 and IL-1 beta, inflammatory mediators which are thought to play a role in the destruction of the periodontium . The purpose of this study was to determine the effect of ST alone and in combination with a major stimulator of inflammation, bacterial lipopolysaccharide (LPS), on monocyte secretion of these mediators . Peripheral blood monocytes (PBM) were isolated by counterflow centrifugal elutriation from 15 healthy donors who were non-ST users . PBM were incubated for 24 hours in RPMI 1640 containing various concentrations of ST (0%, 0.005%, 0.01%, 1%) with or without 10 micrograms/ml LPS (Porphyromonas gingivalis LPS or Escherichia coli LPS) . Of the ST preparations, only 1% ST resulted in PBM mediator secretion (7.7 +/- 2.0 ng/ml for PGE2 and 1.3 +/- 0.2 ng/ml for IL-1 beta) above that of control (unstimulated) cultures . Furthermore, the combination of 1% ST and LPS resulted in a potentiation of PGE2 release (5-fold for E . coli LPS + 1% ST and 10-fold for P . gingivalis LPS + 1% ST; P < 0.0001, one-way ANOVA) relative to the LPS preparations alone . In contrast, PBM IL-1 beta release decreased more than 2-fold upon E . coli LPS and 1% ST exposure, relative to treatment with E . coli LPS alone (P < 0.0001, one-way ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)

Biophys J, 1994 Oct, 67(4), 1682 - 90
Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N; Tittor J et al.; Proton translocation activity of bacteriorhodopsin mutants lacking the proton acceptor Asp-85 was investigated using the black lipid membrane technique . Mutants D85N, D85T, and D85,96N were constructed and homologously expressed in Halobacterium salinarium to yield a membrane fraction with a buoyant density of 1.18 g/cm3, i.e., identical to that of wild-type purple membrane . In all mutants, the absorbance maximum was red-shifted between 27 and 49 nm compared with wild type, and the pKa values of the respective Schiff bases were reduced to between 8.3 and 8.9 compared with the value of > 13 in wild type . Therefore, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) exists at physiological pH . In continuous blue light, the deprotonated form generates stationary photocurrents . The currents are enhanced by a factor of up to 50 upon addition of azide in D85N and D85,96N mutants, whereas D85T shows no azide effect . The direction of these currents is the same as in wild type in yellow light . Yellow light alone is not sufficient to generate stationary currents in the mutants, but increasing yellow light intensity in the presence of blue light leads to an inversion of the current . Because all currents are carried by protons, this two-photon process demonstrates an inverted proton translocation by BR mutants.

Biophys J, 1994 Oct, 67(4), 1546 - 61
Effect of charged residue substitutions on the thermodynamics of signal peptide-lipid interactions for the Escherichia coli LamB signal sequence; Jones JD et al.; We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles . These peptides harbor charged residue substitutions in the hydrophobic core region . Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively . We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide . However, the difference between wild-type and mutant peptide affinities was much lower under approximately physiological ionic strength . In addition, the lipid affinities of model surface-binding and transmembrane peptides were determined . These comparative studies with signal and model peptides permitted semi-quantitative deconvolution of signal peptide binding into electrostatic and hydrophobic components . We find that both interactions contribute significantly to binding, although the theoretically available hydrophobic free energy is largely offset by unfavorable polar-group effects . The implications of these results for understanding the potential roles of the signal sequence in protein translocation are discussed.

Biophys J, 1994 Oct, 67(4), 1534 - 45
Effect of charged residue substitutions on the membrane-interactive properties of signal sequences of the Escherichia coli LamB protein; Jones JD et al.; Although the central role of the signal sequence in protein export is well established, the molecular details underlying signal sequence in vivo function remain unclear . As part of our continuing effort to relate signal sequence phenotypes to specific biophysical properties, we have carried out an extensive characterization of the secondary structure and lipid interactions for a family of peptides corresponding to the wild-type E . coli LamB signal sequence, and mutants that harbor charged residue point mutations in the hydrophobic core region . We used membrane-resident fluorescence quenching according to the parallax method to determine the relative depth of insertion of tryptophan-labeled analogs of these peptides into the acyl chain region of bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol . Also, restriction of acyl chain motion upon peptide binding was evaluated using steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene . Each of these peptides showed evidence of insertion into the acyl chain region, although most likely not in a transmembrane orientation . The mutant peptides were shown to have a reduced insertion potential relative to the wild-type peptide . Furthermore, tryptophan spectral properties indicated that insertion of the wild-type and mutant peptides enhances bilayer hydration . This effect was particularly pronounced with peptides harboring negatively charged aspartate point substitutions . The results are discussed in relation to the potential roles of signal sequences in mediating protein translocation.

Pediatr Nephrol, 1994 Oct, 8(5), 545 - 7
Racial incidence of hemolytic uremic syndrome; Jernigan SM et al.; Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children and is caused by infection with verotoxin-producing Escherichia coli . There is no consensus on the relative incidence of HUS in blacks and whites . An equal racial incidence has been reported by two centers with small black populations . A series from Washington D.C . reported a low incidence in blacks . The population of Alabama is 32% black and 66% white . The Children's Hospital of Alabama admission rate has a similar racial distribution (35% black, 65% white) . A record review from 1980-1992 identified 45 patients with HUS; 43 (96%) were white and only 2 (4%) were black . Based on census data for Alabama in 1980 and 1990, this gives an average annual incidence of HUS of 0.45 per 100,000 in whites and of 0.043 per 100,000 in blacks (P < 0.001, Fischer's exact test) . Similar results were found in the group of patients with HUS and a history of diarrhea; whites 0.39 and blacks 0.02 (P < 0.001) . However, in those with no history of diarrhea there was no significant racial difference: whites 0.05 and blacks 0.02 . There were too few blacks to compare clinical course and outcome . We conclude that typical diarrhea-associated HUS is a relatively rare disease in blacks compared with whites . The reasons are unclear.

Trends Biochem Sci, 1994 Oct, 19(10), 426 - 7
Methods and reagents . Better competent cells and DNA polymerase contaminants; Hengen PN; Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methods-reagents, available on the Internet . This month's column discusses a new method for harvesting Escherichia coli cells for transformation, and contaminants found in DNA polymerase stocks used for the polymerase chain reaction . For details on how to partake in the newsgroup, see the accompanying box.

Poult Sci, 1994 Oct, 73(10), 1534 - 41
Parental effect on the humoral immune response to Escherichia coli and Newcastle disease virus in young broiler chicks; Leitner G et al.; Genetic and environmental variables influence animal resistance to disease infection . In addition, maternal effects were also found in studies with egg-type chicken lines . In our laboratory, meat-type chicken lines were divergently selected for either early or late maturation of the immune system, based on family and individual antibody responsiveness at 10 d of age . The high-antibody (HC) and low-antibody (LC) lines differed significantly in the early immune response to Escherichia coli, to Newcastle disease virus (NDV) vaccination, and to several other immune functions . Reciprocal crosses between the HC and LC lines were performed over 2 yr at three different locations . Immune responses to E . coli and NDV vaccination provided separate estimates of maternal and paternal effects . Dam effect on immune response to E . coli vaccine was significantly larger than sire effect; the antibody titer in both reciprocal crosses was intermediate between the parental lines, but the mean titer of the HC x LC cross was significantly lower than that of the LC x HC cross . Similar, but not significant, ranking of crosses was observed for the response to NDV . Evidently, the level of the offspring humoral immune response was more a dam than a sire effect.

Biometals, 1994 Oct, 7(4), 292 - 8
Site-directed mutagenesis of the ferric uptake regulation gene of Escherichia coli; Coy M et al.; The 12 histidine and four cysteine residues of the Fur repressor of Escherichia coli were changed, respectively, to leucine and serine by site-directed mutagenesis of the fur gene . The affects of these mutations were measured in vivo by ligation of the mutated genes to a wild-type fur promoter followed by measurement of the ability of these plasmids to regulate expression of a lacZ fusion in the aerobactin operon . In vitro affects were assayed by insertion of the mutated genes in the expression vector pMON2064 attended by isolation of the altered Fur proteins and appraisal of their capacity to bind to operator DNA . The results suggest that cysteine residues at positions 92 and 95 are important for the activity of the Fur protein.

Scand J Clin Lab Invest, 1994 Oct, 54(6), 465 - 73
Lipoproteins do not modulate the tissue factor activity, plasminogen activator or tumour necrosis factor production induced by lipopolysaccharide stimulation of human monocytes; Schlichting E et al.; Upon stimulation with lipopolysaccharides (LPS), monocytes are able to produce tissue factor (TF), the most powerful physiological procoagulant substance known . In several assay systems LPS bound to lipoprotein has been reported to be less active than unbound LPS in stimulating monocytes . In the present study the LPS-induced TF activity was, however, not prevented by lipoproteins (VLDL, LDL, HDL) . In fact, the very low density (VLDL) fraction further increased the TF inducing capacity of LPS . The lipoproteins per se mediated reduced plasminogen activator (PA) production in monocytes . LPS had an even more and significant depressing effect on PA production, which was not further decreased in the presence of lipoproteins . Furthermore, LPS-induced release of tumour necrosis factor (TNF), a marker of monocyte activity, was not inhibited by lipoproteins . Our experiments suggest that lipoproteins do not render LPS less effective in stimulating TNF release, procoagulant and fibrinolytic activities in human monocytes.

Oral Microbiol Immunol, 1994 Oct, 9(5), 305 - 9
Homology among Treponema denticola plasmids; Reedy E et al.; Three of 16 isolates of Treponema denticola were found to contain small (2.0-2.7 kb) cryptic plasmids . These were pTD1 from T . denticola ATCC 33520, pTD2 from strain T32A, and pTD3 from strain D3A1 . These plasmids were characterized by restriction mapping and cloned into E . coli plasmid pUC19 . Extensive homology between these plasmids was revealed by Southern blot, and single-stranded DNA regions were found by neutral Southern blots and S1 nuclease mapping . These plasmids were not found in serovars usually associated with human periodontal disease nor are they universal in all T . denticola strains and serovars.

Neuropeptides, 1994 Oct, 27(4), 235 - 44
Prohormone convertases PC2 and PC3 in rat neutrophils and macrophages . Parallel changes with proenkephalin-derived peptides induced by LPS in vivo; Vindrola O et al.; Prohormone- or proneuropeptide-converting enzymes PC2 and PC3 have been observed exclusively in nervous and endocrine tissues . In this work the presence of these enzymes in cells of the immune system was demonstrated . PC2 was detected in peripheral and liver-infiltrating polymorphonuclear leukocytes (PMN) but not in alveolar macrophages (AM) or spleen mononuclear cells (SMC) . PC2 proteins corresponded to 75, 71 and 56 kDa forms . PC3 appeared in AM and SMC but not in PMN, and a 66 kDa protein was the only PC3 form detected . Proenkephalin-derived peptides (PENKp) were observed in PMN and AM, showing peptides of 35, 28, 21, 18 and 14 kDa in the former cells and a doublet of 35 and 32 kDa in the latter . PC2 proteins and PENKp decreased in liver PMN and peripheral PMN 90 min after intravenous (i.v.) infusion of LPS, suggesting an increased release . However, in vitro assays showed that the chemotactic peptide FMLP but not LPS increased the basal secretion of PC2 proteins and PENKp in PMN . These results indicate that PC2 proteins are released from PMN, together with PENKp, and suggest that LPS in vivo may act through an indirect mechanism . Low levels of PC3 and PENK were detected in the AM of rats treated for 90 min with SAL or LPS . However, a significant increase of PC3 and PENKp appeared 30 h after LPS infusion . These results show for the first time that PC2 and PC3 are differentially expressed in PMN and AM, respectively, which were paralleled by the presence of different post-translational products of PENK . In addition, the in vivo effect of LPS on PC2, PC3 and PENKp levels in PMN and AM resembles the effect of LPS on prohormone levels in endocrine tissues, suggesting that similar mechanisms may control the turnover of PENK in endocrine and in these immune cells.

Yakugaku Zasshi, 1994 Oct, 114(10), 747 - 64
{Synthesis and functional studies on nucleic acids . Problems related to synthetic ras genes}; Ohtsuka E; Synthesis of a gene for an oncoprotein, ras p21 and its functions are described . Point mutations in hot spots of ras genes have been found in human cancer cells and produce activated p21 which result in transformation of cultured NIH3T3 cells . To produce normal and activated p21 in quantity for biochemical and structural studies, genes encoding these proteins were synthesized and expressed in E . coli . Normal and activated RAS proteins were tested for their GTPase activity and three dimensional structures were determined by X-ray crystallography . Transforming activities of the synthetic genes have been tested by transfecting their expression vectors to NIH3T3 cells and the synthetic activated genes were found to transform these cells indicating that the product of the activated gene is responsible for these malignant growth of the cells . These activities were proved to be inhibited by transfecting designed ribozyme genes . These synthetic genes were used to investigate mutagenesis of damaged bases such as 7,8-dihydro-8-oxoguanine and thymine photodimers, by introducing the damaged base in hot spots of the oncogene . These unnatural bases in the ras gene were found to be mutagenic and cause malignant growth of NIH3T3 cells.

Bioorg Med Chem, 1994 Oct, 2(10), 1085 - 90
Alpha-ketoamide Phe-Pro isostere as a new core structure for the inhibition of HIV protease; Munoz B et al.; Studies on the inhibition of HIV-1 protease utilizing a core isostere with replacement of the scissle bond for an alpha-amino-ketone have resulted in the development of an alpha-keto-amide isosteric replacement of the Phe-Pro scissle amide bond . The simple dipeptide isostere was shown to be a promising new core structure for the development of the enzyme inhibitors . The Ki of this core structure was determined to be 6 microM, compared to 230 microM and > 50 microM for the corresponding phosphinic acid and hydroxyethylamine isosteres.

Appl Microbiol Biotechnol, 1994 Oct, 42(1), 73 - 7
Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV; Oswald T et al.; The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated . N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase {GST}, histidine hexapeptide {(His)6}, or a combination of GST and (His)6 {GST-(His)6} were compared to native EcoRV with respect to expression level, susceptibility to inclusion body formation and protein fragmentation . Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non-equilibrium pH-gradient electrophoresis with subsequent immunoblotting . Fusion proteins containing GST were poorly expressed compared to native EcoRV . In addition, GST fusion proteins were highly susceptible to in-vivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots . However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous . Fragmentation of (His)6-EcoRV was not observed and 2-D immunoblots did not show heterogeneous forms of the recombinant protein . In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV . Inclusion body formation of the (His)6-EcoRV fusion protein was intensive when cells were grown at 37 degrees C but not at 30 degrees C . The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)6-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1918 - 9
Histamine synthesis by bone marrow-derived macrophages; Takamatsu S et al.; Histamine may be involved in the regulation of hematopoiesis . However, it remained obscure what kind of cells are responsible for its synthesis in bone marrow (BM) . In this study, a pure population of macrophages were raised from BM of W/Wv mice, which are genetically deficient in mast cells . The addition of Escherichia coli lipopolysaccharide (LPS) or murine recombinant granulocyte/macrophage colony-stimulating factor (mrGM-CSF) alone had essentially no effect on histamine synthesis . However, the latter rendered the cells responsive to LPS, leading to a marked increase in HDC-dependent histamine synthesis.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1899 - 901
A host-vector system for a cellulose-producing Acetobacter strain; Tonouchi N et al.; An indigenous plasmid, named pAH4, was detected in a cellulose-producing Acetobacter strain . This plasmid, consisting of 4002 bp, contained an AT-rich region and encoded several open reading frames, as deduced by the complete nucleotide sequence . One of the putative open reading frames showed homology with replication proteins of other plasmids . A shuttle vector of Escherichia coli and this strain was constructed by connecting pAH4 to pUC18 . Electroporation of the shuttle vector into the strain yielded 1.7 x 10(5) ampicillin resistant transformants per microgram DNA . The shuttle plasmid was very stably maintained in the strain.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1814 - 9
Evidence that Escherichia coli ubiA product is a functional homolog of yeast COQ2, and the regulation of ubiA gene expression; Suzuki K et al.; The Escherichia coli ubiA gene coding for 4-hydroxy benzoate octaprenyl transferase is thought to be a key enzyme of ubiquinone biosynthesis . Strains with ubiA disrupted were constructed by chromosomal gene replacement with the chloramphenicol resistance gene . The respiration-defective phenotype of the ubiA mutant was complemented by expression of the COQ2 gene encoding the 4-hydroxy benzoate hexaprenyl transferase of Saccharomyces cerevisiae and such strains produced ubiquinone-8 . This strongly supports the idea that COQ2 catalyzes the same enzymatic reaction with UbiA and the substrate specificity of COQ2 is broad . Study of the expression of ubiA using an ubiA-lacZ fusion system showed that the ubiA expression was catabolite-repressed by glucose . This repression by glucose was obvious in the arcA mutant . ArcA is the positively acting transcriptional regulator of the oxygen regulated genes . The molecular mass of the protein product of ubiA was 32kD, found using the over-expression of the ubiA gene.

Bioseparation, 1994 Oct, 4(5), 319 - 28
Disruption of a recombinant yeast for the release of beta-galactosidase; Garrido F et al.; A recombinant yeast, Saccharomyces cerevisiae, expressing Escherichia coli beta-galactosidase gene under the control of CYC1 constitutive promoter of the yeast, was disrupted in a continuous flow, high speed, bead mill for the release of intracellular beta-galactosidase (EC 3.2.1.23) . Release of the beta-galactosidase activity was characterized with respect to glass bead loading in the grinding chamber (70-85% of chamber volume), diameter of the beads (0.25-0.75 mm), number of passes of the cell slurry through the mill (0-6 passes), flow rate of the slurry (25-250 mL.min-1), cell concentration in the slurry (5-20 gDW.L-1), the agitation rotor speed (1000-4000 rpm) and the pH of the slurry (pH 5-10) . The optimal conditions for the release of the enzyme were pH 6.0-9.0, 85% loading of 0.5 mm diameter beads and an agitation speed of 2000 rpm . The enzyme release followed first-order kinetics . For otherwise fixed conditions, the extent of cell disruption increase with increasing bead load, number of passes and agitation rotor speed . Cell concentration did not affect disruption . The release of beta-galactosidase activity declined with increasing flow rate of the cell slurry through the mill, but the disruption rate constant increased with flow rate . Under optimal condition, three passes through the grinding chamber were sufficient to release all of the enzyme . In comparison with disruption in the bead mill, chloroform-sodium dodecyl sulfate induced lysis of cells was ineffective in releasing the enzyme quantitatively.

Bioseparation, 1994 Oct, 4(5), 299 - 309
Toxicity studies on Reactive Blue-2 leached from affinity material exposed to extreme chemical conditions; Bertrand O et al.; Toxicity effects related to leached ligands from affinity sorbents that can contaminate biological preparations were investigated in the particular case of immobilized Reactive Blue-2 . Initially, identification of the real chemical structure of leached dye has been done by HPLC after incubation in extreme conditions . Toxicity investigations in vitro involving several well known tests showed no toxic effects within the studied range of dye concentration . Cell cultures behaved normally when the adhesion phase was successful; polyploidy induction in human cells by the native dye and its derivatives identified as possible leached material was very similar to standard cultures . Genotoxicity studies did not evidence any toxic effect in E . Coli cultures of dyes themselves or of the same dyes after metabolic activation.

Curr Opin Biotechnol, 1994 Oct, 5(5), 534 - 9
Protein chaperones and protein folding; Gilbert HF; A universal strategy for obtaining maximal protein expression or refolding remains elusive; however, headway has been made toward understanding these processes in vivo . The observation of reversible protein aggregation, asymmetry in protein-chaperone complexes, redox effects on disulfide formation, and the sequential involvement of multiple chaperones and foldases may suggest new approaches . Such new approaches include immobilized catalysts and manipulation of the bacterial periplasm.

Curr Opin Biotechnol, 1994 Oct, 5(5), 487 - 94
Construction and screening of biological peptide libraries; Schatz PJ; Significant advances have been made recently in technology to construct and screen peptide libraries using biological systems . Progress has been achieved in increasing the size of libraries, in controlling affinity of the peptides isolated, and in understanding the constraints imposed by the biology of the expression systems employed . New receptor ligands and substrates for peptide-modifying enzymes have been isolated using these powerful techniques.

Curr Opin Biotechnol, 1994 Oct, 5(5), 482 - 6
Applications of interaction traps/two-hybrid systems to biotechnology research; Mendelsohn AR et al.; Two-hybrid methods provide a simple and sensitive means to detect the interaction between two proteins in living cells . Their use has resulted in the isolation of new proteins and has facilitated characterization of particular protein-protein interactions . These techniques have already resulted in the identification of important targets for pharmaceutical intervention, and it is likely that their extension in coming years will facilitate the development of new drugs.

Shock, 1994 Oct, 2(4), 289 - 95
Increased surface expression of CD11b/c and CD18 appears to be dissociated from anti-CD11b/c monoclonal antibody stimulated O2- anion generation in in vivo Escherichia coli lipopolysaccharide and tumor necrosis factor-alpha-treated rat neutrophils; Mayer AM et al.; The purpose of this investigation was to determine the effect of anti-rat CD11b/c monoclonal antibody (MAb) on in vitro superoxide anion (O2-) generation in in vivo Escherichia coli lipopolysaccharide (LPS) and tumor necrosis-alpha (TNF)-treated rat polymorphonuclear leukocytes (PMN) . After a continuous infusion of a nonlethal dose of E . coli LPS (.5 mg/kg) or TNF (6.0 x 10(5) units) into rats, PMN were recovered by centrifugal elutriation and discontinuous density gradient centrifugation from the liver (LPS-treated) and whole blood (LPS- and TNF-treated) and compared for CD11b/c, CD11a, and CD18 upregulation and their capacity for basal and agonist-stimulated O2- production . Immunofluorescence flow cytometry studies of rat whole blood PMN demonstrated that, upon LPS infusion, there was a time-dependent upregulation of CD11b/c and CD18, but not of CD11a . Similarly, TNF infusion upregulated CD11b/c although to a lesser degree . Stimulation of LPS- and TNF-treated PMN with phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OPZ), and anti-rat CD11b/c MAb triggered O2- generation . Although total O2- generated by OPZ and anti-rat CD11b/c MAb was less than that generated by PMA stimulation, the in vivo LPS- and TNF-induced beta 2 integrin upregulation did not result in a statistically significant enhancement of O2- generation with respect to normal saline-treated PMN . Our results do not appear to support the hypothesis that enhanced expression of CD11b/c or CD18 might be associated with enhanced in vitro anti-CD11b/c MAb-triggered O2- generation in LPS- and TNF-treated PMN in vivo.

Shock, 1994 Oct, 2(4), 275 - 80
Beneficial effects of a novel platelet-activating factor receptor antagonist, ST 899, on endotoxin-induced shock in mice; Ruggiero V et al.; The effect of ST 899, a novel platelet-activating factor (PAF) receptor antagonist, on serum tumor necrosis factor (TNF), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma) production as well as on lethality in an experimental endotoxin shock model was investigated in C57BL/6 mice . In this model, animals receiving 40 mg/kg lipopolysaccharide (LPS) (Escherichia coli 055:B5) intraperitoneally were pretreated with ST 899 administered according to two different schedules . ST 899 pretreatment dose dependently reduced the mortality induced by LPS injection . The PAF receptor antagonist was also able to reduce significantly the LPS-induced increase in serum TNF . Although serum IL-6 levels were not affected, we found that ST 899, when administered intraperitoneally 60 min and intravenously 10 min prior to LPS challenge, had a tendency (at higher doses) to decrease circulating IFN-gamma levels . It is suggested that ST 899 may be beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of PAF, TNF, and IFN-gamma such as septic shock.

Shock, 1994 Oct, 2(4), 251 - 6
The Kupffer cell in endotoxin tolerance: mechanisms of protection against lethal endotoxemia; Hafenrichter DG et al.; Kupffer cells (KC) of the hepatic sinusoid respond to endotoxemia by producing mediators which promote or inhibit systemic inflammatory responses . Sublethal lipopolysaccharide (LPS) pretreatment confers tolerance to the lethality of a subsequent LPS exposure . However, the precise role of the KC in endotoxin tolerance (ET) remains unclear . This study evaluated the effect of ET induction upon the rat KC production of the mediators tumor necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2), and interleukin-6 (IL-6), and upon the in vivo phagocytic capacity of the KCs . 3 days prior to KC isolation, age-matched rats received either 5 mg/kg LPS (ET) or normal saline (nontolerant, NT), which protected 100% of the ET rats against an LPS dose 3 days later which was lethal in 72% of NT rats . On an in vitro LPS rechallenge, ET KC produced significantly lower amounts of TNF than NT KC (p < .01) . In contrast, the ET KC produced significantly more PGE2 (p < .05) and IL-6 (p < .001) than the NT KC . The percentage of KC phagocytosing fluorescent latex spheres in vivo was increased 7-fold in the ET rats . Thus, ET induction, which protects rats against subsequent lethal endotoxemia, selectively alters KC mediator production and phagocytic capacity . These findings strongly implicate the KC in the mediation of early endotoxin tolerance.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1994 Oct, 16(5), 387 - 9
{IL-1 beta mRNA expression in patients with rheumatoid arthritis}; Yu M et al.; Using arthroscopy techniques, 6 synovia samples from patients with rheumatic diseases were obtained . Of these, 2 patients had rheumatoid arthritis (RA), 2 had meniscus damage (MD), 1 had osteoarthritis (OA), and 1 had osteochondritis (OC) . Using the guanidinium isothiocyanate method, total RNA from synovia was extracted, and 18S and 28S sedimentation bands (rRNA) were separated by electrophoresis . Plasmid containing the 1.3kb cDNA of IL-1 beta were extracted from E . coli by alkaline lysis . Subsequent digestion with XHO1 and then separation on gel isolated the 1.3kb cDNA for use as a probe . The probe was then labeled with 32P-dATP by nicktranslation and hybridized to the synovial RNA extract . The autoradiograph showed that in 4 cases of non-RA, the IL-1 beta cDNA did not hybridize to the synovial mRNA extract . An intense hybridization band was visible for mRNA from one RA case . The clinical and laboratory parameters and X-rays of both knees and wrists of this case were all abnormal, and a rthroscopic findings showed pathological rheumatoid changes of synovium . In addition, this patient did not receive any DMARDs treatment for the RA . The other RA case did not have any hybridization bands . However, this patient did receive DMARDs treatment, and the RA went into remission . The Northern blot result above suggests that IL-1 beta mRNA expression in synovia is negative in non-RA cases and in cases where RA is in remission after DMARDs treatment . In the case with clinically active RA, IL-1 beta is highly expressed, as evidenced by the intense hybridization band.

Microb Pathog, 1994 Oct, 17(4), 261 - 70
Introduction of plasmid carrying an incomplete set of genes for aerobactin production alters virulence of Escherichia coli HB101; Harjai K et al.; The aerobactin-mediated iron uptake system is encoded by pColV-K30 and other ColV plasmids . It has been known to contribute to the ability of Escherichia coli to cause pyelonephritis and cystitis . In the present study an attempt was made to evaluate the contribution of an incomplete set of genes for aerobactin synthesis to the virulence of Escherichia coli HB101 . Escherichia coli HB101 was transformed with a recombinant plasmid pJHCV-12 (Tetr and Kanr) carrying aerobactin genes (complete first two genes, iucA and iucB and part of the third gene iucC) from pColV-K30 . Both HB101 and a transformant H10 grew equally well when applied to a Vero cell line . These strains were tested for their ability to invade and kill Vero cells in monolayers . Light micrographs showed cell damage by the transformant carrying pJHCV-12 plasmid and this cytotoxic effect correlated with the amount of lactate dehydrogenase (LDH) released . In contrast, strain HB101 and HB101 containing parent vector pVK102 did not produce any cytotoxic effects . When the ability of these strains to produce ascending pyelonephritis in a mouse model was compared, the transformant established itself better in renal tissue than the control strain HB101, when assessed 2h, 4 h and 5 days post-infection.

Avian Dis, 1994 Oct-Dec, 38(4), 708 - 16
Effect of infection with hemorrhagic enteritis virus on susceptibility of turkeys to Escherichia coli; van den Hurk J et al.; The present study was designed to investigate hemorrhagic enteritis virus (HEV) as a predisposing factor influencing the susceptibility of young turkeys to Escherichia coli infections . In addition, the pathologic changes caused by administration of E . coli by various routes were compared . Following oral infection with HEV, groups of turkeys were inoculated with various doses of pathogenic E . coli by intravenous (IV), intra-air sac (IA), or intratracheal (IT) routes . A synergistic effect was observed in birds that were exposed to a combined HEV-E . coli challenge, resulting in higher mortality than that caused by either pathogen alone . This synergy was more evident when the bacteria were administered by the IT route than when it was administered by the two other routes . Turkeys infected with HEV and then inoculated IT with E . coli O78 had higher mortality (61%) and higher occurrence of gross body lesions (74%) than birds given E . coli alone (0% mortality and 16% gross lesions) . After E . coli inoculation by the IA and IT routes, lesions observed were mainly pericarditis, perihepatitis, lung and air-sac lesions, splenic enlargement, and occasional arthritis . The incidence of lesions was affected by HEV exposure . In contrast, IV inoculation with E . coli O78 usually resulted in arthritis, and its incidence was independent of previous HEV exposure . The synergistic effect between HEV and E . coli administered IT can be used as a challenge model for testing E . coli vaccines.

Braz J Med Biol Res, 1994 Oct, 27(10), 2383 - 9
Membrane permeability and sensitivity to lethal heat are affected by lexA and recA mutations in Escherichia coli K12; Lage C et al.; Membrane permeability was evaluated in several SOS-deficient strains . Great heat sensitivity was observed in all the lexA (Ind-) strains, which was associated to an increase in membrane permeability (up to 120% increase above the wild-type control), as assayed by the crystal violet (CV) growth inhibition . After irradiation with a single UV dose (75 J.m-2 delivered to wild-type and 2 J.m-2 to the lexA3 strain), survival was followed by plating cells in both nutrient and membrane permeability-selective (nutrient + CV) media and a great lethality due to CV was observed in a lexA mutant, which appeared to be about 100 times more sensitive to CV compared to its wild-type parent strain . The decreased membrane integrity found in the lexA-deficient strains suggests that LexA protein and/or LexA-repressed genes may interact with the bacterial membrane, which could be the location of SOS events.

Can J Vet Res, 1994 Oct, 58(4), 302 - 5
Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping; Harel J et al.; This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9 . For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis . For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates . Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S . hyodysenteriae isolates were observed . REF and ribotyping using BglII restriction enzyme, were not specific for serotypes . The predominance of an REF and a ribotype pattern among S . hyodysenteriae isolates from Quebec suggested that epidemiologically important S . hyodysenteriae types occur in different swine herds.

Am J Reprod Immunol, 1994 Oct, 32(3), 194 - 9
Decay-accelerating factor is expressed in the human endometrium and may serve as the attachment ligand for Dr pili of Escherichia coli; Kaul A et al.; PROBLEM: We evaluated the hypothesis that different tissue substructures in uteri may express decay accelerating factor (DAF), a complement regulatory protein that also may serve as ligand for bacterial attachment . METHOD: Purified Dr pili, anti-Dr pili IgG, anti-DAF (SCR-3) IgG, and fluorescein-isothiocyanate-conjugated secondary IgG were used for binding and inhibition experiments . RESULT: We observed staining of endometrial glands, spiral arterioles, and myometrial arteries with Dr adhesin (pili) and anti-DAF (SCR-3) IgG, and found variation in distribution and amount of Dr ligands in different individuals . Anti-DAF (SCR-3) IgG blocked the binding of Dr pili to the endometrium . CONCLUSION: Presence of DAF in endometrium may protect tissues from complement-induced damage . Differences between individuals in DAF density in the endometrium may affect sensitivity to attachment of Dr-bearing E . coli and/or complement activation.

Anal Biochem, 1994 Oct, 222(1), 102 - 9
Subtraction hybridization cloning of RNA amplified from different cell populations microdissected from cryostat tissue sections; Luqmani YA et al.; We describe a generally applicable and easily reproducible method for the cloning of differentially expressed RNA, amplified from small numbers of enriched cell populations, obtained by microdissection from single cryostat sections . The procedure involves homopolymeric A tailing of cDNA synthesized from released RNA using an anchored (NN)T12 primer . Subsequent entire cDNA population polymerase chain reaction amplification was carried out using a biotinylated (X)nT16 primer-adaptor in the presence of biotin-dATP . This biotinylated driver cDNA was then twice hybridized in 50-fold excess to heterologous target cDNA made with nonbiotinylated (Y)nT16 primer; common hybrids and excess driver cDNA were magnetically removed following the addition of streptavidin-coated magnetospheres which bound biotinylated strands, leaving enriched target population sequences . These were then directly amplified through the tails using a primer containing only the target-specific (Y)n sequence . Insertion into a lambda-phage vector was facilitated by means of an EcoR1 site incorporated in the (Y)n primer . Subsequent packaging and transformation into Escherichia coli NM522 resulted in cDNA libraries containing approximately 5 x 10(3)-10(4) pfu . Screening of these primary libraries with cDNA derived from the starting populations yielded a large number of differentially hybridizing clones which are currently under analysis.

Brain Res Mol Brain Res, 1994 Oct, 26(1-2), 277 - 85
Rapid and stable gene expression in hippocampal slice cultures from a defective HSV-1 vector; Bahr BA et al.; Stable transfer of genetic information into neurons is a powerful strategy to elucidate specific mechanisms of neurophysiology and to develop therapies for neurological disorders . To evaluate the optimal parameters for efficient gene delivery of defective herpes simplex virus type one (HSV-1) vectors into a specific brain region, an HSV-1 vector expressing E . coli beta-galactosidase was used to infect organotypic cultures of hippocampal slices . beta-Galactosidase was expressed as early as 2 h after infection in a dose-dependent manner as measured on immunoblots, and reached a maximum level after approximately 35 h . Expression of the RNA and the antigen was still evident after the longest time sampled (11-12 days), whereas no beta-galactosidase was ever detected in cultured slices infected with a control virus lacking the reporter gene . Hippocampal cells expressing the reporter gene outlined the contour of the neuronal cell body layers in fields CA3 and dentate gyrus; such correspondence was less evident in field CA1 . Anatomical, morphological, and immunohistochemical criteria also confirmed that the majority of these infected cells were neurons . beta-Galactosidase was also detected in the somata and processes of infected interneurons . Tests for synaptic pathology associated with virus infection showed no changes in pre- and postsynaptic markers.

Biotechniques, 1994 Oct, 17(4), 754 - 61
An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments; Knappik A et al.; The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity . Using the calcium-dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD) . Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods . Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp . The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E . coli is processed correctly . Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position . Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins.

Intern Med, 1994 Oct, 33(10), 641 - 3
Drug eruption caused by recombinant human G-CSF; Sasaki O et al.; Two types of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are available, and equally used for mitigation of neutropenia . One is a glycosylated natural product from mammalian cells, and the other a non-glycosylated form from Escherichia coli . Though only minimal adverse effects have been reported for both, we treated two patients with rhG-CSF-induced systemic eruption . Based on these patients, the following should be noted: 1) drug eruption may occur in both types of rhG-CSF without detectable antibodies, 2) intradermal test is useful for determination of the causal drug, and 3) if one rhG-CSF product causes eruption, the alternative one may possibly be safe and effective.

Am J Physiol, 1994 Oct, 267(4 Pt 2), R1020 - 5
Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes; Williams G et al.; Nitric oxide (NO) is an important mediator of diverse physiological and pathological responses . To determine whether NO production can be induced in skeletal muscle, we stimulated C2C12 mouse skeletal muscle myocytes with putative inducers of nitric oxide synthase (NOS) . Neither lipopolysaccharide (LPS), interleukin-1 alpha (IL-1), tumor necrosis factor-alpha (TNF), nor interferon-gamma (IFN) was able to stimulate nitrite production by C2C12 cells when administered alone . However, combinations of IFN with either TNF or IL-1 resulted in significant nitrite production; simultaneous stimulation of cells with all three cytokines resulted in significantly increased nitrite production compared with any combination of two cytokines . Northern analysis of RNA obtained from stimulated C2C12 cells revealed induction of a single mRNA band that precisely coincided with the mRNA band of mouse macrophage-inducible NOS (iNOS) . Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis followed by sequencing of the 5' 765 bases of the skeletal muscle iNOS cDNA demonstrated exact homology with mouse macrophage iNOS . These findings indicate that combinations of cytokines stimulate NO production in skeletal muscle cells via induction of the macrophage-type iNOS gene.

J Bacteriol, 1994 Oct, 176(19), 6030 - 8
Nucleoid partitioning and the division plane in Escherichia coli; Woldringh CL et al.; Escherichia coli nucleoids were visualized after the DNA of OsO4-fixed but hydrated cells was stained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate) . In slowly growing cells, the nucleoids are rod shaped and seem to move along the major cell axis, whereas in rapidly growing, wider cells they consist of two- to four-lobed structures that often appear to advance along axes lying perpendicular or oblique to the major axis of the cell . To test the idea that the increase in cell diameter following nutritional shift-up is caused by the increased amount of DNA in the nucleoid, the cells were subjected to DNA synthesis inhibition . In the absence of DNA replication, the nucleoids continued to move in the growing filaments and were pulled apart into small domains along the length of the cell . When these cells were then transferred to a richer medium, their diameters increased, especially in the region enclosing the nucleoid . It thus appears that the nucleoid motive force does not depend on DNA synthesis and that cell diameter is determined not by the amount of DNA per chromosome but rather by the synthetic activity surrounding the nucleoid . Under the non-steady-state but balanced growth conditions induced by thymine limitation, nucleoids become separated into small lobules, often lying in asymmetric configurations along the cell periphery, and oblique and asymmetric division planes occur in more than half of the constricting cells . We suggest that such irregular DNA movement affects both the angle of the division plane and its position.

J Bacteriol, 1994 Oct, 176(19), 5999 - 6006
Mutations in the -10 TATAAT sequence of the gyrA promoter affect both promoter strength and sensitivity to DNA supercoiling; Straney R et al.; Transcription of the gyrA and gyrB genes, which encode the subunits of DNA gyrase, increases in response to DNA relaxation . Previous studies have shown that a small segment of DNA extending from the -10 consensus hexamer to the start of transcription encodes the sequence determinants for this response . In this study, we examined the role of the -10 region in relaxation-stimulated transcription (RST) . A synthetic derivative of the gyrA promoter was designed to allow the modular replacement of the -10 region, and mixed-oligonucleotide mutagenesis was used to obtain a collection of promoter mutants . Most substitutions result in a reduction of the promoter's RST response, and some mutations abolish it altogether . We also note that a variety of promoter changes can increase basal expression twofold above that seen for the gyrA promoter, despite sequences changes (up to three bases) which diverge from the consensus TATAAT of the wild-type gyrA hexamer . The wild-type gyrA promoter, however, is the strongest promoter in this collection on a relaxed DNA template and appears to be repressed on a supercoiled template in vivo . Our results are consistent with a mechanism for RST that involves a step in transcription initiation.

Infect Immun, 1994 Oct, 62(10), 4611 - 7
Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells; Konig B et al.; The Escherichia coli alpha-hemolysin represents a potent stimulus for inflammatory mediator release (O2-, beta-glucuronidase release, and leukotriene generation) from human polymorphonuclear granulocytes, for histamine release from a suspension of human lymphocyte/monocyte basophil cells (LMB), and for serotonin release and 12-hydroxyeicosatetraenoic acid generation from human platelets . In contrast, the E . coli alpha-hemolysin leads to a downregulation of cytokine release (interleukin-1 beta {IL-1 beta}, IL-6, and tumor necrosis factor alpha) from human LMB . Recently, it became apparent that the E . coli alpha-hemolysin is composed of several functional structures . We analyzed the role of pore formation, pore stability, and calcium-dependent membrane binding for inflammatory mediator release by using washed bacteria as well as culture supernatants of isogenic recombinant E . coli strains expressing no hemolysin (Hly-), the wild-type hemolysin (Hly+), or hemolysin molecules deficient or modulated in defined functions (pore formation, calcium-dependent membrane binding, or pore stability) . In human granulocytes and platelets, mutant hemolysin with enhanced pore stability did not lead to a further increase in induction; mutant hemolysin deficient in pore-forming activity or calcium-dependent membrane binding no longer induced leukotriene B4 generation or beta-glucuronidase release compared with the wild-type hemolysin . Similar results were obtained with regard to histamine release from human LMB . The induction of cytokine release from human LMB differed depending on the type of mutant E . coli alpha-hemolysin . The wild-type hemolysin, the mutant hemolysin with enhanced pore-forming activity, and, to a lesser degree, the mutant hemolysin deficient in pore-forming activity decreased cytokine release (IL-1 beta, IL-6, IL-8, and tumor necrosis factor) compared with untreated cells . In contrast, the mutant hemolysin deficient in calcium-dependent membrane binding led to an increase of up to 50% in cytokine release compared with that by unstimulated cells . Our results indicate that simultaneous expression of the pore-forming and calcium-dependent membrane-binding activities of the hemolysin molecule was necessary to obtain the full cellular inflammatory response pattern observed with the wild-type hemolysin.

Clin Exp Immunol, 1994 Oct, 98(1), 104 - 9
Mapping of B cell epitopes on steroid 17 alpha-hydroxylase, an autoantigen in autoimmune polyglandular syndrome type I; Peterson P et al.; Earlier studies have shown that 17 alpha-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc) are the main autoantigens recognized by sera from patients with Addison's disease associated with autoimmune polyglandular syndrome type I (APS I) . In this study we tried to identify the autoantigenic epitopes on P450c17 and compared the identified sequences with corresponding regions in two other adrenal autoantigens, P450scc and P450c21 . A series of P450c17 cDNA fragments was expressed in Escherichia coli and the recognition of the corresponding protein fragments by patient sera was tested by immunoblotting . Four distinct epitope regions (ER) were found: ER1 (amino acids 122-148), ER2 (280-304), ER3 (396-432) and ER4 (466-508) . B cell epitope prediction analysis showed that the four identified ERs were all located in regions of high predicted antigenicity . Homology search revealed that ER3 and ER4 but not ER1 and ER2 were related to similar sequences on P450c21 . No significant homologies with P450scc in these regions were found . Although all three P450 cytochromes are genuine autoantigens this finding suggests that the autoantibody reaction against P450c17 and P450c21 can partly be a result of immunological cross-reactivity.

Virology, 1994 Oct, 204(1), 466 - 70
Expression of the glycoprotein H of murine cytomegalovirus and identification of an N-terminal antibody-binding region; Xu J et al.; A series of overlapping fragments spanning the entire coding sequence of the gH gene of murine cytomegalovirus (MCMV) were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system . A region of antibody-binding was mapped to the NH2-terminus of glycoprotein H (gH) between amino acid residues 26 and 90 on the basis of the reactivity of GST-gH fusion proteins with polyclonal antibodies to MCMV in Western blot analysis . Antibodies to gH were generated in mice immunized with the GST-gH fusion protein SK and shown to react with an 87-kDa polypeptide present in virion particles which was conserved in MCMV isolates obtained from diverse locations . They also recognized the gH protein in MCMV-infected cells, as well as gH expressed in Chinese Hamster Ovary cells . The antibodies to gH had a significant ELISA titer but no neutralizing activity in vitro . The antibody response to the GST-gH fusion protein did not modify the level of MCMV replication in the spleens of mice.

Virology, 1994 Oct, 204(1), 254 - 65
Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein; Rouleau M et al.; The open reading frame 2 (ORF2) of the potexviral genome encodes a 24- to 26-kDa protein which is part of the "triple gene block," a group of overlapping ORFs also present in the genomes of the carla-, hordei-, and furoviruses . The product of these ORFs is believed to play a role in the cell-to-cell movement of the viruses in host plants . The amino acid sequences of the homologous ORF2 products encoded by these related viruses suggest that they specify NTP binding and possibly helicase activities . We have used an Escherichia coli expression system to produce significant amounts of the 26-kDa protein (p26) encoded by foxtail mosaic potexvirus ORF2 . p28 was purified to near homogeneity by conventional purification methods and some of its biochemical properties were determined . We present evidence that p26 is an ATP, CTP, and RNA binding protein with apparent ATPase activity . Western blot analysis of infected plant extracts using a polyclonal antiserum produced against p26 indicates that it is a relatively stable protein maintained at high levels for at least 6 days following its peak level of expression . Moreover, it is found predominantly in the soluble fraction of infected tissues . An immunocytochemical analysis of infected Chenopodium quinoa leaves reveals that p26 is exclusively associated with cytoplasmic inclusions in proximity to but distinct from aggregates of viral particles.

J Mol Biol, 1994 Sep 30, 242(4), 582 - 5
Crystallization of GreA, a transcript cleavage factor from Escherichia coli; Darst SA et al.; GreA is a 17.6 kDa protein from Escherichia coli that induces cleavage of the nascent transcript in the elongating complex of RNA polymerase, followed by release of the 3'-terminal fragment . Crystals of GreA have been obtained from polyethylene glycol 4000, 2-propanol and sodium citrate, pH 5.6 and have been propagated by a novel seeding procedure . The crystals diffract beyond 2 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 101.7 A, b = 42.22 A, c = 40.05 A and with one molecule in the asymmetric unit.

J Mol Biol, 1994 Sep 30, 242(4), 566 - 76
Methionyl-tRNA synthetase needs an intact and mobile 332KMSKS336 motif in catalysis of methionyl adenylate formation; Schmitt E et al.; The family of aminoacyl-tRNA synthetases may be split into two classes according to the occurrence of specific combinations of peptide motifs . This study deals with the functional role of the KMSKS motif, which, in association with the HIGH motif, defines class 1 aminoacyl-tRNA synthetases . Each residue in the 332KMSKS336 sequence of Escherichia coli methionyl-tRNA synthetase, as well as R337 and the two surrounding G330 and G338 residues, were mutagenized . The comparison of the kinetic and equilibrium parameters of the methionine activation reaction sustained by the resulting variants enables the following conclusions to be drawn . (1) Mutation of all the residues studied strongly destabilizes the transition state for the formation of methionyl adenylate whilst changing moderately the stability of the ground state ternary complex enzyme, methionine: ATP-Mg2+ . The consequences of the mutations are also reflected at the level of the stability of the ground state enzyme, methionyl adenylate:PPi-Mg2+ complex which is systematically decreased . (2) The substitution with alanine of any one of the three basic residues K332, K335 and R337 destabilizes the transition state by more than 3.2 kcal/mol, while substitution of the non-basic residues M333, S334 or S336 destabilizes it by at most 2.5 kcal/mol . Such a difference may reflect different modes of action of the residues, with the basic ones directly interacting with the beta and gamma phosphates of the ATP-Mg2+ substrate and the non-basic ones playing a structural role and/or participating in mobility of the enzyme region carrying the motif . (3) Modification of G330 or G338 to a proline markedly decreases the kinetic rate of methionyl adenylate formation . This behaviour suggests that the flexibility of the KMSKS loop in the structure of methionyl-tRNA synthetase is required to reach the transition state during formation of methionyl adenylate.

J Mol Biol, 1994 Sep 30, 242(4), 408 - 21
ATP synthase from bovine heart mitochondria . In vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence; Collinson IR et al.; Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli . They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d . Likewise, fragments b', bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment d' (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression . These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase . Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase . When fragments b', bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1.OSCP.b', F1.OSCP.bI or F1.OSCP.bC were formed, but b', bI and bC appeared to be present in sub-stoichiometric amounts . When F6 was added also, then the stoichiometric quaternary complexes F1.OSCP.b'.F6 and F1.OSCP.bI.F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6 . Finally, three pentameric complexes F1.OSCP.b'.F6.d, F1.OSCP.b'.F6.d' and F1.OSCP.b.F6.d were isolated . In a further series of reconstitution experiments, the binary complexes b'.OSCP and b'.d, the ternary complex b'.d'.F6, and the quaternary complex OSCP.b'.F6.d were obtained . The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase . It was shown by S-carboxymethylation of cysteine residues with iodo-{2-14C}acetic acid that bovine F1F0-ATPase and the reconstituted F1.stalk complex, F1.OSCP.b'.d.F6, each contained one copy per complex of subunits b (or b'), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1:1:1 . The ratio of b to d in purified F0 was 1:1 . Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold . These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.

J Mol Biol, 1994 Sep 30, 242(4), 378 - 88
Interaction of EcoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5'-CAGCAG-3'; Ahmad I et al.; EcoP15I DNA methyltransferase (Mtase) recognizes the asymmeteric sequence CAGCAG and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the second adenine residue . We have investigated the DNA binding properties of EcoP15I DNA Mtase using gel mobility shift assays . EcoP15I DNA Mtase binds approximately threefold more tightly to DNA containing its recognition sequence, CAGCAG, than to non-specific sequences in the absence or presence of cofactors . Interestingly, in the presence of ATP the discrimination between specific and non-specific sequences increases significantly . These results suggest for the first time a role for ATP in DNA recognition by type III restriction-modification enzymes . In addition, we have shown that bromodeoxyuridine-containing oligonucleotides form complexes with EcoP15I DNA Mtase that are crosslinked upon irradiation . More importantly, we have shown that the crosslink site is at the site of DNA binding, since it can be suppressed by an excess of unmodified oligonucleotide . EcoP15I DNA Mtase exhibited Michaelis-Menten kinetics with both unmodified and bromodeoxyuridine-substituted DNA, with a higher specificity constant for the latter . Furthermore, gel mobility shift assays showed that proteolyzed EcoP15I DNA Mtase formed a specific complex with DNA, which had similar mobility as the native protein-DNA complex . Taken together these results form the basis for a detailed structure-function analysis of EcoP15I DNA Mtase.

J Mol Biol, 1994 Sep 30, 242(4), 339 - 50
Induction of the SOS response by IS1 transposase; Lane D et al.; We find that IS1 transposase, like that of Tn10, can induce the SOS response when produced at high levels . Most of the activity (> 80%) requires IS1 ends in cis to the transposase gene and depends strictly on the presence of RecBCD function . This implies that processing of transposase-induced cleavages is responsible for generating the response . Induction of the SOS response during growth in a rich medium is seen only when cells approach stationary phase . The end-dependent induction is abolished by mutations in the ends of IS1 that eliminate transposition activity . IS1 ends in identical orientation on the same plasmid are inactive in transposition but stimulate SOS strongly . Even plasmids with a single end can stimulate SOS, probably as a consequence of plasmid dimer formation which places the ends in direct repeat orientation . These results imply that transposase-induced cleavages do not need inversely oriented ends . The system can therefore be used to dissociate cleavage activity from the other reactions of transposition . Induction of SOS by a series of short (67 to 114 bp) IS1-like elements was found to occur in a cyclical pattern as a function of length with a period of 10 to 11 bp . The frequency of cointegration promoted by these elements showed the same helix-phase dependence . These results suggest that transposase molecules bound to the ends of IS1 interact, and that this interaction is needed for the cleavages that initiate transposition.

J Mol Biol, 1994 Sep 30, 242(4), 330 - 8
Reaching out . Locating and lengthening the interdomain linker in AraC protein; Eustance RJ et al.; A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions . Attractive candidates for these areas are linker regions . Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory protein AraC . Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain . Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177 . The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case . Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site . In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA . In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.

J Biol Chem, 1994 Sep 30, 269(39), 24430 - 6
Amino-terminal conserved region in proteinase inhibitor domain of calpastatin potentiates its calpain inhibitory activity by interacting with calmodulin-like domain of the proteinase; Ma H et al.; Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca(2+)-dependent proteinase) and is known to interact with the calmodulin-like domain (CaMLD) of the proteinase in a Ca(2+)-dependent fashion . The calpastatin molecule consists of four inhibitory domains (domains 1-4) with mutually homologous sequences in three regions designated as A, B, and C . Acidic amphiphilic alpha-helical motifs are found in both regions A and C . We investigated the correlation between the calpain inhibition potency and the ability of calpastatin to bind to recombinant CaMLD of the mu-calpain large subunit using various mutant proteins of pig calpastatin domain 1 expressed in Escherichia coli . Substitution of conserved Leu-161 with Pro in region A caused a reduction in activity of both calpain inhibition and CaMLD binding . Additional substitution of Leu-236 with Pro in region C further decreased the calpain inhibitory activity and caused a loss of CaMLD binding ability . The effects of mutation in region C alone on the above activities were smaller than those in region A . Although a mutant of deletion in the entire region B had no calpain inhibitory activity, it retained the CaMLD binding ability . On the other hand, although a region B oligopeptide had a moderate inhibitory activity, it had no CaMLD binding ability . These results suggest that region A has a role in potentiating the inhibitory activity of calpastatin by interacting with CaMLD of calpain to form a tighter complex where region B exerts the inhibitory function.

J Biol Chem, 1994 Sep 30, 269(39), 24374 - 8
2 cloning and expression of mouse deoxycytidine kinase . Pure recombinant mouse and human enzymes show differences in substrate specificity; Karlsson A et al.; A cDNA encoding mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) was cloned from a mouse T-cell lambda ZAP cDNA library . An insert of 2.8 kilobases (kb) contained the entire coding sequence of 780 base pairs . The protein coding sequence was 88% homologous at the nucleotide level with human dCK cDNA (Chottiner, E . G., Shewach, D . S., Datta, N . S., Ashcraft, E., Gribbin, D., Ginsburg, D., Fox, I . H., and Mitchell, B . S . (1991) Proc . Natl . Acad . Sci . U . S . A . 88, 1531-1535) . At the amino acid level the homology was greater with only 16 of the 260 amino acids being different . Northern blot analyses revealed a size of 3.4 kb for mouse dCK mRNA as compared with 2.8 kb for human dCK . Part of the 3'-untranslated region was conserved between human and mouse dCK cDNA in contrast to the remainder of the 3'-sequence which was unrelated and about 500 nucleotides longer in mouse dCK cDNA . Mouse dCK cDNA showed cross-hybridization with several bands in EcoRI-digested genomic DNA from seven different mammalian species and chicken but not with yeast DNA . Both mouse and human dCK were cloned into the T5 promotor pQE30 vector system, expressed in Escherichia coli and purified to homogeneity . The kinetic constants for dCyd phosphorylation were similar for the human and mouse enzymes and also similar to what previously has been observed for dCK purified from human tissues . Mouse dCK was less efficient with regard to dAdo, dGuo, and ddCyd phosphorylation as compared with human dCK when using ATP as phosphate donor in a phosphoryl transfer assay.

J Biol Chem, 1994 Sep 30, 269(39), 24271 - 6
Functional domain structure of human centromere protein B . Implication of the internal and C-terminal self-association domains in centromeric heterochromatin condensation; Sugimoto K et al.; Centromere protein B (CENP-B) is a common centromere DNA-binding protein among mammalian centromeres . CENP-B possesses the specific DNA binding activity to the 17-base pair sequence dispersed in centromeric repetitive DNA sequences . In the previous study, we have shown that its DNA-binding domain exists within the N-terminal 134 amino acid residues . Here, to clarify the whole domain structure, another functional unit required for CENP-B self-association was examined . Recombinant CENP-B was expressed in Escherichia coli . First, a chemical cross-linking reagent, disuccinimidyl suberate, was used to fix the physical association without losing the DNA-binding activity . The complexes with the same molecular weight as homodimer and trimer were identified after a separation by SDS-polyacrylamide gel electrophoresis . With a series of CENP-B deletion constructs, the area responsible for this oligomer formation was located at the internal region . Second, an electrophoretic mobility shift assay was also used to survey the minimum regions required for the CENP-B self-association . Three separate elements were identified by assaying the capacity to form the additional slow-migrating complex, two in the internal region and one in the C terminus . These results suggest that CENP-B molecules interact with each other at the multiple sites to fold the centromeric DNA repeats into a heterochromatin structure.

J Biol Chem, 1994 Sep 30, 269(39), 24066 - 72
Mutagenesis of amino acid residues required for binding of corepressors to the purine repressor; Choi KY et al.; The corepressor-binding domain of the Escherichia coli purine repressor (PurR) is homologous with several periplasmic sugar-binding proteins . Four amino acids in PurR were investigated for a role in binding of corepressors . Three of the residues, Asp146, Arg196 and Asp275, are conserved in periplasmic binding proteins for ribose, glucose/galactose, and arabinose and function to bind sugars . A fourth amino acid, Trp147, required for corepressor binding to PurR, corresponds to residues in glucose/galactose, ribose, and arabinose that also have a role in sugar binding . The four mutations that were constructed perturbed the binding of both hypoxanthine and guanine thus providing evidence for a single corepressor site/PurR subunit . The decreased corepressor binding affinity resulted in reduced affinity of mutant repressors for operator DNA in vitro and decreased capacity for repression in vivo . The corepressor-binding site in PurR appears to be similar to the conserved ligand-binding sites in the three periplasmic sugar-binding proteins and in the LacI family of repressors.

J Biol Chem, 1994 Sep 30, 269(39), 24046 - 9
Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5'-triphosphate to xanthosine 5'-triphosphate; Kang C et al.; The aspartate residue of the (N/T)KXD concensus sequence for GTP-binding proteins is present in the eight available sequences of adenylosuccinate synthetase . Reported here is a comprehensive analysis of the substrate specificity of mutant enzymes, where the conserved Asp333 of the synthetase from Escherichia coli is changed to asparagine, glutamate, and glutamine by site-directed mutagenesis . The mutants D333N, D333E, and D333Q generally show decreased kcat values and increased Km values for GTP . The decreased values of kcat exhibited by the mutants indicate that the interactions between Asp333 and the guanine are relayed by some mechanism to the catalytic residues around the gamma-phosphate of GTP, and that the energy provided by the interaction between Asp333 and the guanine moiety of GTP is utilized for rearrangement of the catalytic residues . The three mutants each have higher affinity for xanthosine 5'-triphosphate (XTP) and ITP than does the wild-type enzyme . In fact, the D333N mutant uses XTP more effectively than the wild-type enzyme employs GTP as a substrate . The side-chain of Asp333 forms hydrogen bonds with the N-1 and the exocyclic amino group of the guanine base of GTP . In the D333N mutant, this interaction is probably replaced by hydrogen bonds between the amide side chain of Asn333 and N-1 and the 2-oxo group of XTP . The D333Q mutant can use UTP as a substrate more effectively than the wild-type enzyme . The longer side chain of glutamine at residue 333 favors pyrimidine nucleotides over the purine nucleotides, GTP, XTP, and ITP . These results demonstrate that Asp333 in the (N/T)KXD consensus sequence of adenylosuccinate synthetase from E . coli is a determinant for GTP-specificity.

J Biol Chem, 1994 Sep 30, 269(39), 23869 - 71
ATP induces non-identity of two rings in chaperonin GroEL; Bochkareva ES et al.; For its function, the Escherichia coli chaperonin GroEL requires the presence of ATP and co-chaperonin GroES . We have observed that ADP displays a two-step inhibition of GroEL-dependent ATP hydrolysis, wherein one-half of the GroEL ATPase sites is strongly inhibited by ADP while the other half is affected very mildly . It is suggested that interaction with ATP induces structural and functional differences between two initially identical rings in GroEL (inter-ring negative cooperativity) and that the subsequent binding of GroES occurs to the ring that is occupied first by ATP in a positively cooperative manner.

Gene, 1994 Sep 30, 147(2), 281 - 5
Cloning of a human cDNA encoding a putative nucleotide-binding protein related to Escherichia coli MinD; Shahrestanifar M et al.; A novel human cDNA encoding a putative nucleotide-binding protein (NBP) was obtained by screening a human SHSY5Y neuroblastoma library . The deduced protein contains 320 amino acids (aa) with a M(r) of 34,540 . NBP displays sequence similarity with the product of the minD gene from Escherichia coli . MinD is involved in the proper placement of the division septum, and has ATPase activity . NBP and MinD contain consensus nucleotide (nt)-binding domains . The NBP mRNA is approx . 1500 nt in length and is expressed in several human cell lines and in all rat tissues examined, with the highest levels in lung and testis.

J Biotechnol, 1994 Sep 30, 37(2), 133 - 42
Expression of a neutral horseradish peroxidase in Escherichia coli; Bartonek-Roxa E et al.; A cDNA sequence encoding a neutral horseradish peroxidase, HRP-n, was found to be growth inhibiting and under certain circumstances also toxic to Escherichia coli upon expression . The growth inhibiting and toxic activity was identified to a sequence of 192 nucleotides which encode the first deduced 64 N-terminal amino acids of the total 299 amino acids in the mature, neutral horseradish peroxidase . The sequence makes part of the active site of the enzyme and is very conserved among peroxidases . The cDNA sequence was cloned in the heat inducible expression vector pJLA603 . The toxic effect was mediated by the produced polypeptide since no growth inhibiting or toxic activity was observed when the cDNA sequence was induced after ligation in a wrong reading frame . Generation of oxygen radicals was not the mechanism behind the toxicity, since the effect was observed even after induction of the complete HRP-n sequence or the identified toxic sequence under anaerobic conditions.

J Biol Chem, 1994 Sep 30, 269(39), 24277 - 83
A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope; Raben N et al.; In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase . To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system . It was enzymatically active and recognized by human autoantibodies . A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme . This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase . Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not . The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so . The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

Nature, 1994 Sep 29, 371(6496), 432 - 5
XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair; O'Donovan A et al.; Humans with a defect in the XPG protein suffer from xeroderma pigmentosum (XP) resulting from an inability to perform DNA nucleotide excision repair properly . Here we show that XPG makes a structure-specific endonucleolytic incision in a synthetic DNA substrate containing a duplex region and single-stranded arms . One strand of the duplex is cleaved at the border with single-stranded DNA . A cut with the same polarity is also made in a bubble structure, at the 3' side of the centrally unpaired region . Normal cell extracts introduce a nick 3' to a platinum-DNA lesion, but an XP-G cell extract is defective in making this incision . These data show that XPG has a direct role in making one of the incisions required to excise a damaged oligonucleotide, by cleaving 3' to DNA damage during nucleotide excision repair.

Nature, 1994 Sep 29, 371(6496), 429 - 32
Interaction between two homeodomain proteins is specified by a short C-terminal tail; Stark MR et al.; Two yeast homeodomain proteins, a1 and alpha 2, interact and cooperatively bind the haploid-specific gene (hsg) operator, resulting in the repression of a set of genes involved in the determination of cell type . The cooperative binding of a1 and alpha 2 to DNA can be reconstituted in vitro using purified fragments of a1 and alpha 2 . Only the homeodomain is needed for a1, but for alpha 2 a C-terminal 22-amino-acid tail is required as well . As most of the specificity of DNA binding appears to derive from a1, we proposed that alpha 2 functions in the a1/alpha 2 heterodimer to contact a1 with its tail . By construction and analysis of several chimaeric proteins, we investigate how two DNA-binding proteins, one with low intrinsic specificity (alpha 2) and one with no apparent intrinsic DNA-binding ability (a1), can together create a highly specific DNA-binding activity . We show that the 22-amino-acid region of alpha 2 immediately C-terminal to the homeodomain, when grafted onto the a1 homeodomain, converts a1 to a strong DNA-binding protein . This alpha 2 tail can also be attached to the Drosophila engrailed homeodomain, and the chimaeric protein now binds cooperatively to DNA with a1, showing how a simple change can create a new homeodomain combination that specifically recognizes a new DNA operator.

Nature, 1994 Sep 29, 371(6496), 423 - 6
A brain-specific activator of cyclin-dependent kinase 5; Lew J et al.; Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity . We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau . The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref . 8) . When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro . Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain . A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract . Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.

Biochim Biophys Acta, 1994 Sep 29, 1223(3), 341 - 7
Gain of function mutations for yeast calmodulin and calcium dependent regulation of protein kinase activity; Lukas TJ et al.; Yeast calmodulin binds only three calcium ions in the presence of millimolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain . Yeast calmodulin's diminished calcium-binding activity can be restored to that of other calmodulins by the use of site-directed mutagenesis to substitute its fourth calcium-binding domain with that of a vertebrate calmodulin sequence . However, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective protein kinase activator activity . Yeast calmodulin's activator activity with smooth muscle and skeletal muscle myosin light chain kinases and brain calmodulin-dependent protein kinase II can be progressively repaired by additional substitutions of vertebrate calmodulin sequences, provided that the four calcium-binding sites remain intact . An unexpected result obtained during the course of these studies was the observation that myosin light chain kinases from smooth and skeletal muscle tissues can respond differently to mutations in calmodulin . These and previous results indicate that the binding of four calcium ions by calmodulin is necessary but not sufficient to bring about quantitative activation of protein kinases, and are consistent with the conformational selection/restriction model of the dynamic equilibrium among calcium, calmodulin and each calmodulin regulated enzyme.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9617 - 21
A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation; Hong Z et al.; Phosphatidylinositol 3-kinase (PI3K) is an important component of various receptor tyrosine kinase complexes in mammalian cells and a key enzyme required for cell division and vacuolar protein sorting in yeast . To our knowledge, this enzyme has not been characterized in plants . We report the cloning and characterization of soybean PI3K cDNAs and present evidence for the induction of a distinctive form of this enzyme specific to nodule organogenesis . Expression of the root form of PI3K is repressed during nodule organogenesis and is reinduced in mature nodules . Primer-extension results showed that the gene encoding the nodule form of PI3K is highly expressed in young (12-15 day old) root nodules in parallel with membrane proliferation but is repressed in mature nodules . The root form of the PI3K cDNA (SPI3K-5) encodes a peptide of 814 amino acids and the nodule form (SPI3K-1) encodes a peptide of 812 amino acids . Both cDNAs share 98% sequence identity in the coding region but differ in the noncoding regions . The polypeptides encoded by soybean PI3K cDNAs show significant sequence homology (50-60% similarity and 20-40% identity) to both PI3Ks and phosphatidylinositol 4-kinases from mammalian and yeast cells . Escherichia coli expressed soybean PI3K phosphorylated phosphatidylinositol specifically at the D-3 position of the inositol ring to generate phosphatidylinositol 3-phosphate . The temporal increase of a specific PI3K activity during membrane proliferation in young nodules suggests that PI3K plays a pivotal role in development of the peribacteroid membrane forming a subcellular compartment.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9342 - 6
Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity; Harth G et al.; We have investigated the activity and extracellular release of glutamine synthetase {L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2} of Mycobacterium tuberculosis . The purified, homogeneous M . tuberculosis glutamine synthetase appears to consist of 12 most likely identical subunits of M(r) 58,000, arranged in two superimpose hexagons . In the catalysis of L-glutamine, the enzyme has an apparent Km for L-glutamate of approximately 3 mM at the pH optimum of 7.5 . M . tuberculosis releases a large proportion (approximately 30%) of its total measurable enzyme activity into the culture medium, a feature that is highly specific for pathogenic mycobacteria . Immunogold electron microscopy revealed that M . tuberculosis also releases the enzyme into its phagosome in infected human monocytes . Two potentially important roles for glutamine synthase in the pathogenesis of M . tuberculosis infection are (i) the synthesis of L-glutamine, a major component of the cell wall of pathogenic but not nonpathogenic mycobacteria, and (ii) the modulation of the ammonia level in the M . tuberculosis phagosome, which may in turn influence phagosomal pH and phagosomelysosome fusion.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9302 - 6
Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter; Furth PA et al.; Promoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo . We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice . In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence . Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer . Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold . This induction was abrogated to basal levels upon administration of tetracycline . These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.

Biochim Biophys Acta, 1994 Sep 27, 1187(3), 354 - 9
Over-production, renaturation and reconstitution of delta and epsilon subunits from chloroplast and cyanobacterial F1; Steinemann D et al.; We studied the functioning of chimeric F0F1-ATPases by replacing subunits delta and epsilon of spinach CF1 with their counterparts from Synechocystis sp . PCC 6803 . The sequence identities between these subunits are 26 and 41%, respectively . For a systematic approach to such studies and later extension to genetically modified subunits recombinant proteins are required . The genes coding for spinach and Synechocystis delta and epsilon were cloned into pET3 expression vectors and expressed in Escherichia coli . Upon expression at 37 degrees C the recombinant subunits formed inclusion bodies within the host cells except for spinach delta, which was soluble . Synechocystis delta and epsilon could be obtained in soluble form upon expression at 20 degrees C . After purification (and refolding of spinach epsilon) both epsilon subunits inhibited the Ca(2+)-ATPase activity of soluble CF1(- epsilon) . Subunits delta and epsilon from both species raised the rate of ATP synthesis in partially CF1-depleted spinach thylakoids when added together with CF1(- delta) or CF1(- delta, epsilon) . This showed the functionality of recombinant Synechocystis and spinach delta and epsilon together with spinach alpha 3 beta 3 gamma . The molar excess of epsilon necessary for saturation was higher for Ca(2+)-ATPase inhibition than for reconstitution of photophosphorylation thus pointing to a direct interaction between epsilon and both CF1 and CF0.

Biochemistry, 1994 Sep 27, 33(38), 11664 - 70
Functional immunoliposomes harboring a biosynthetically lipid-tagged single-chain antibody; Laukkanen ML et al.; An anti-2-phenyloxazolone single-chain antibody was expressed in Escherichia coli as a lipoprotein fusion in order to generate a biosynthetically lipid-tagged molecule {Laukkanen et al . (1993) Protein Eng . 6, 449-454} . For purification, a hexahistidinyl tag was introduced to the C-terminus of the protein . The resulting antibody, termed Ox lpp-scFv-H6, was membrane-bound, displayed hapten-binding activity, and contained the lipoprotein-specific lipid modification as indicated by metabolic {3H}palmitic acid labeling . The Ox lpp-scFv-H6 was purified by immobilized metal affinity chromatography followed by hapten-based affinity chromatography to essential homogeneity with a yield of 0.4-1.6 mg/L of culture . In detergent dialysis, the purified antibody partitioned quantitatively into phospholipid liposomes . The immunoliposome preparation consisting of a homogeneous population of unilamellar 100-200 nm vesicles displayed specific hapten-binding activity as measured by using ELISA and surface plasmon resonance (SPR)-based real-time biospecific interaction analysis . In SPR experiments, the immunoliposomes exhibited virtually irreversible binding to immobilized hapten compared to soluble antibody fragments, consistent with the predicted multivalent binding . Biosynthetic lipid-tagging of antibodies may prove useful for immunoliposome-based diagnostic and therapeutic applications.

Biochemistry, 1994 Sep 27, 33(38), 11624 - 30
Probing of the retinal binding site of bacteriorhodopsin by affinity labeling; Feng Y et al.; The position of the chromophore within bacteriorhodopsin has been identified by cross-linking a cysteine group, introduced by site-specific mutagenesis, with a chromophore suitably derivatized with an active leaving group . Since bacteriorhodopsin has no cysteines, a site-specific cysteine mutant will contain only one free sulfhydryl group capable of reacting with the retinal analog . Met118, Thr121, and Ser141 were selected to be mutated to cysteine . No pigment absorbing in the visible region was obtained for the Ser141Cys mutant . The Met118Cys and Thr121Cys mutants have similar absorption maxima, proton pumping efficiencies and photocycles to those of the wild-type pigment . 4-Bromoretinal, in which the reactive allylic halide readily undergoes nucleophilic displacement, was used as the reactive chromophore . Pigments were obtained on reaction of all-trans-4-bromoretinal with the apoproteins of Met118Cys, Thr121Cys, and wild-type bacteriorhodopsin (lambda max = 464-470 nm) . Analysis of the denatured pigments on SDS-polyacrylamide gels showed incorporation of tritiated chromophore into the Met118Cys mutant but not into the wild-type or Th4121Cys pigments . Met118Cys apoprotein which was preincubated with the cysteine-specific reagent N-ethylmaleimide formed a pigment with 4-bromoretinal but no cross-linking was observed, providing evidence that the cross-linking of the chromophore is to the cysteine at 118 . We conclude that Met118 is positioned in the chromophore binding pocket, proximal to the C-4 position of cyclohexyl ring of retinal.

Biochemistry, 1994 Sep 27, 33(38), 11576 - 85
Nonadditivity of mutational effects at the folate binding site of Escherichia coli dihydrofolate reductase; Huang Z et al.; The function of the hydrophobic residues Leu28, Phe31, Ile50, and Leu54 at the folate binding site in Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) has been studied by a combination of site-specific mutagenesis and reaction kinetics . Studies suggest that the overall protein structure and kinetic sequence for the reaction did not change for the mutant proteins compared to the wild-type enzyme . Two sets of mutated reductases have been constructed . The first set, in which the side chains of the targeted amino acids are spatially well separated (approximately 8 A), includes two single mutants (L28Y and L54F) and a double mutant (L28Y-L54F) . This set features residues that increased the side chain surface area and the potential for substrate interactions . Unexpectedly, nonadditivity in the free energy changes for the thermodynamics of ligand binding and in the rates of hydride transfer and product release is observed . The progressive increase in dihydrofolate binding is reversed for the sterically more crowded double mutant, with delta delta G ca . 3 kcal mol-1 less favorable than anticipated . On the other hand, the decrease in the rate constant for hydride transfer noted with the single mutants relative to the wild-type enzyme is reversed for the double mutant, so that delta delta G not equal to is ca . 2 kcal mol-1 more favorable . The second set of mutant proteins includes two double mutants (L28A-F31A and I50A-L54G) in which the selected amino acids are separated by three to four intervening amino acids and a quadruple mutant (L28A-F31A-I50A-L54G) in which the two sets L28A-F31A and I50A-L54G are spatially distinct . This set deleted the side chain surface area to lower the opportunity for substrate interactions . Nonadditivity in the free energy changes associated with key kinetic and thermodynamic parameters is again observed . The decrease in dihydrofolate binding found with the two double mutants is not observed with the quadruple mutant, which binds the substrate with delta delta G ca . 6.5 kcal mol-1 more favorable than expected . Similarly, the quadruple mutant has a larger rate constant for hydride transfer (-delta delta G not equal to congruent to 1.7 kcal mol-1) than predicted . One interpretation for the nonadditivity is that these residues interact through binding of the folate substrate, which serves to link molecularly remote side chain moieties within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1994 Sep 27, 33(38), 11528 - 35
Role of carboxy-terminal region in proofreading function of methionyl-tRNA synthetase in Escherichia coli; Gao W et al.; The synthetic and editing functions of three forms of Escherichia coli methionyl-tRNA synthetase with different C-terminal sequences have been compared in vivo and in vitro . These forms include a full-length wild-type dimer (MRS676), a truncated monomer (MRS547) believed to be equivalent to the biologically active large tryptic fragment, and a third form denoted MRS581* . DNA sequencing revealed that MRS581* is predicted to contain 18 additional amino acids from the wild-type full-length sequence at the carboxy terminus of truncated form MRS547, and this is then fused to an additional 16 amino acids encoded by vector pBR322 . Both MRS676 and MRS581* were found to edit endogenous homocysteine about 20-fold more efficiently than MRS547 in vivo . However, the three methionyl-tRNA synthetases edited exogenously supplied homocysteine in bacterial cultures to similar extents . Purified proteins exhibited no significant differences in editing function in vitro . Synthetic activity of purified MRS676 in vitro was found to be about 2.5-fold higher per subunit compared to the shorter forms of the enzyme . The C-terminal region in E . coli methionyl-tRNA synthetase is thus suggested to play an important role in editing in vivo, most likely by allowing interaction of the enzyme with the methionine biosynthetic pathway . These data support a model of channeling of at least some metabolites in bacterial protein synthesis.

Biochemistry, 1994 Sep 27, 33(38), 11432 - 7
Recombinant human erythrocyte cytochrome b5; Lloyd E et al.; The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990) . Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization . Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein . The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10) . The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9539 - 43
Coupling of RNA displacement and intrinsic termination in transcription from synthetic RNA.DNA bubble duplex constructs; Daube SS et al.; Functional transcription elongation complexes can be formed by adding RNA polymerase in trans to a preformed nucleic acid construct . This construct consists of a double-stranded DNA fragment that contains a noncomplementary (permanent DNA bubble) region into which an RNA primer oligonucleotide has been hybridized . By ligating a DNA fragment containing the strong intrinsic terminator T7Te to the RNA.DNA bubble duplex, we show here that Escherichia coli core RNA polymerase-catalyzed transcription, initiated from such a construct, terminates at the predicted position . Furthermore, we show that the termination efficiency obtained is comparable to that observed in a control reaction initiated with the E . coli holopolymerase from the T7A1 promoter if an RNA oligomer trap is used to permit proper displacement of the nascent RNA from the DNA template strand . The trap oligomer is complementary to the template strand of the permanent DNA bubble and prevents rehybridization of the nascent RNA at this site . Varying the amount of RNA trap that is added permits us to modulate the extent of total RNA displacement . Our results show that RNA displacement and termination efficiency are directly correlated, suggesting that intrinsic termination requires that the nascent RNA be free to assume its solution conformation . Several models of intrinsic termination are presented and discussed in light of these data.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9223 - 7
A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli; Komine Y et al.; We have determined that 10Sa RNA (one of the small stable RNAs found in Escherichia coli) has an interesting structural feature: the 5' end and the 3' end of 10Sa RNA can be arranged in a structure that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E . coli . Primer-extension analysis of 10Sa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to 10Sa RNA (pre-10Sa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA . The purified 10Sa RNA can be charged with alanine in vitro . Disruption of the gene encoding 10Sa RNA (ssrA) caused a reduction in the rate of cell growth, which was especially apparent at 45 degrees C, and a reduction in motility on semisolid agar . These phenotypic characteristics of the deletion strain (delta ssrA) allowed us to investigate the effects of some mutations in 10Sa RNA in vivo, although the exact function of 10Sa RNA still remains unclear . When the G.U pair (G3.U357) in 10Sa RNA, which may be equivalent to the determinant G.U pair of alanine tRNA, was changed to a G.A or G.C pair, the ability to complement the phenotypic mutations of the delta ssrA strain was lost . Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a G.A pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (alaS) on a multicopy plasmid . The evidence suggests that the proposed structural features of 10Sa RNA are indeed manifested in vivo.

FEBS Lett, 1994 Sep 26, 352(2), 243 - 6
N-ethylmaleimide-sensitive mutant (beta Val-153-->Cys) Escherichia coli F1-ATPase: cross-linking of the mutant beta subunit with the alpha subunit; Iwamoto A et al.; A beta subunit mutation, beta Val-153-->Cys, in the glycine-rich sequence (phosphate-binding loop) of Escherichia coli F1 was constructed . Like vacuolar-type ATPase, the mutant enzyme was inhibited by N-ethylmaleimide (NEM) and labeled with {14C}NEM . The inhibition and labeling were prevented by ATP . m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) (3 microM) almost completely inhibited the mutant enzyme, and cross-linked one pair of alpha and beta subunits . These results suggest that the interaction of the domain near beta Val-153 with the alpha subunit is essential for catalytic cooperativity of the enzyme and that beta Val-153 is within 10 A of the alpha subunit.

FEBS Lett, 1994 Sep 26, 352(2), 222 - 6
Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity; Alonso MD et al.; Glycogenin, the self-glucosylating primer for glycogen synthesis, is expressed in wild-type E . coli as a recombinant protein in an already partly glucosylated form, owing to the presence of its substrate, UDP-glucose . By using an E . coli mutant strain lacking in UDP-glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate-free glycogenin (apo-glycogenin) in good yield . When provided with UDPxylose, it autocatalytically adds 1 xylose residue . With UDP-glucose, an average of 8 glucose residues are added . However, release of the self-synthesized maltosaccharide chains with isoamylase reveals them to be a mixture . Chains as long as 11 glucose residues (maltoundecaose) are present . The ability of recombinant apo-glycogenin to self-glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr-194 of the protein.

FEBS Lett, 1994 Sep 26, 352(2), 155 - 8
High level expression and characterisation of Plasmepsin II, an aspartic proteinase from Plasmodium falciparum; Hill J et al.; DNA encoding the last 48 residues of the propart and the whole mature sequence of Plasmepsin II was inserted into the T7 dependent vector pET 3a for expression in E . coli . The resultant product was insoluble but accumulated at approximately 20 mg/l of cell culture . Following solubilisation with urea, the zymogen was refolded and, after purification by ion-exchange chromatography, was autoactivated to generate mature Plasmepsin II . The ability of this enzyme to hydrolyse several chromogenic peptide substrates was examined; despite an overall identity of approximately 35% to human renin, Plasmepsin II was not inhibited significantly by renin inhibitors.

FEBS Lett, 1994 Sep 26, 352(2), 127 - 30
The bacteriochlorophyll biosynthesis gene, bchM, of Rhodobacter sphaeroides encodes S-adenosyl-L-methionine: Mg protoporphyrin IX methyltransferase; Gibson LC et al.; The bchM gene of Rhodobacter sphaeroides has been sequenced and then overexpressed in E . coli producing a protein of M(r) approximately . 27,500 . Cell-free extracts of the transformed E . coli strain are able to methylate added Mg protoporphyrin, resulting in the formation of Mg protoporphyrin monomethyl ester . The identity of this product was verified by HPLC . The bchM gene product is therefore assigned to the methyltransferase step in bacteriochlorophyll biosynthesis.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3930 - 5
5-Hydroxypyrimidine deoxynucleoside triphosphates are more efficiently incorporated into DNA by exonuclease-free Klenow fragment than 8-oxopurine deoxynucleoside triphosphates; Purmal AA et al.; Recent studies with 8-oxodeoxyguanosine triphosphate (8-oxodGTP) have suggested that incorporation of oxidized nucleotides from the precursor pool into DNA may have deleterious effects . Here we show that 5-hydroxydeoxycytosine triphosphate (5-OHdCTP) and 5-hydroxydeoxyuridine triphosphate (5-OHdUTP) are more efficient substrates than 8-oxodGTP for Escherichia coli DNA polymerase I Klenow fragment lacking proofreading activity, while 8-oxodeoxyadenosine triphosphate (8-oxodGTP, 5-OHdCTP can mispair with dA in DNA but with lower efficiency . Since the 5-hydroxypyrimidines are present in normal and oxidized cellular DNA in amounts similar to the 8-oxopurines, these data suggest that enzymatic mechanisms might exist for removing them from the DNA precursor pools.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3880 - 6
Molecular basis of nitrogen mustard effects on transcription processes: role of depurination; Masta A et al.; DNA was alkylated with nitrogen mustard (HN2) and the rate of release of the alkylpurines was quantitated by HPLC . The half life of depurination of the major product (7-alkylguanine) was 9.1 h at 37 degrees C . End-labelled DNA was used to show that depurination occurred dominantly at 5'-GA, 5'-GG and 5'-GT sequences . Although extensive alkylation was observed at all 5'-GNC and 5'GNT sequences, no depurination was observed at these sites during a depurination time of 20 h at 37 degrees C . Since these sites are potential interstrand crosslinking sequences (G-adduct-G and G-adduct-A, both spanning an intervening base pair), this suggests that these regions have a greatly enhanced stability or that simultaneous depurination of both ends of the crosslink is necessary before these lesions are removed (with a predicted half-life of approximately 80 h at 37 degrees C) . Depurination at the lac UV5 promoter impaired the association of Escherichia coli RNA polymerase with that promoter, while in the elongation phase two distinctly different sequence-specific processes were apparent . At 5'-GNC and 5'-GNT sequences transcriptional blockages were maintained with increasing elongation time, whereas at monoadduct sites, the blockage decreased with elongation time (predominantly at 5'-GG and 5'-GC sequences), with an average half-life of approximately 10.7 h . Collectively, these results suggest that the observed read-through past monoadduct sites is due to depurination of the DNA at those sites . E . coli RNA polymerase is therefore able to transcribe efficiently past apurinic sites and presumably does so by incorporating an incorrect base into the nascent RNA.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3871 - 9
A residue of the ETS domain mutated in the v-ets oncogene is essential for the DNA-binding and transactivating properties of the ETS-1 and ETS-2 proteins; Soudant N et al.; The c-ets-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that recognize purine-rich motifs . The v-ets oncogene of the avian retrovirus E26 differs from its cellular progenitor p68c-ets-1 by two amino acid substitutions (alanine 285 and isoleucine 445 in c-ets-1 both substituted by valine in v-ets, mutations A and B respectively) and its carboxy-terminal end (mutation C) . The B mutation affects a well conserved residue in the carboxy-terminal 85 amino acids, ETS DNA-binding domain . To address the biological relevance of the B mutation found between v-ets and c-ets-1, we have randomly mutagenized isoleucine 445 of p68c-ets-1 by polymerase chain reaction . Using in vitro gel mobility shift assays, we show that this residue is crucial for the binding properties of c-ets-1 since the 12 mutations we have generated at this position, all diminish or even abolish the binding, to the 'optimized' Ets-1 binding site (EBS), of 35 kDa proteins corresponding to the 311 carboxy-terminal residues of c-ets-1 . Among them, substitutions of isoleucine to glutamic acid, glycine or proline have the highest inhibitory effects . Similar results were obtained when the same mutations were introduced either in full-length p68c-ets-1 protein or into a carboxy-terminal polypeptide of 109 amino acids encompassing the ETS-domain which has previously been shown to display a very high binding activity as compared with the full-length protein . Consistent with the in vitro results, point mutations in p68c-ets-1 that decrease binding activity to EBS abrogate its ability to transactivate reporter plasmids carrying either the TPA Oncogene Response Unit of the Polyoma virus enhancer (TORU) or a sequence derived from the HTLV-1 LTR . Furthermore, as this isoleucine residue is rather well-conserved within the ETS gene family, we show that mutation of the corresponding isoleucine of c-ets-2 into glycine also abrogates its DNA-binding and hence, transactivating properties . Thus, the v-ets B mutation highlights the isoleucine 445 as an essential amino acid of the c-ets-1 and c-ets-2 DNA-binding domains.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3840 - 5
Thermal energy requirement for strand separation during transcription initiation: the effect of supercoiling and extended protein DNA contacts; Burns H et al.; We have studied the role of extended protein DNA contacts and DNA topology on the ability of Escherichia coli RNA polymerase to form open complexes at several related promoters . The -35 region of several Escherichia coli promoters do not have homology with the consensus sequence, but still drive activator independent transcription initiation . This is due to the presence of a TG motif upstream from the -10 hexamer creating an 'extended -10' promoter . We have previously shown that two 'extended -10' promoters, galP1 and pBla, can form open complexes at lower temperatures than the galP1 derivative, galPcon6, which has a consensus -35 hexamer . Here we report further investigations into the mechanism of open complex formation by RNA polymerase, in particular the thermal energy requirement . A single base pair change in galPcon6 creating an 'extended -10' sequence, results in a 20 degrees C reduction in the temperature requirement for open complex formation . The DNA topology has also been shown to effect the thermal energy requirement for strand separation . Promoters carried on supercoiled plasmids form open complexes at lower temperatures than when present on linear DNA templates . We have also shown that in vivo, RNA polymerase can form open complexes at lower temperatures than those observed for linear templates in vitro, but requires slightly higher temperatures than supercoiled templates in vitro, however the promoter hierachy remains the same.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3819 - 24
Efficient targeting of foreign genes into the tobacco plastid genome; Zoubenko OV et al.; The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA) . The vectors are based on the pUC119 plasmid which replicates in E . coli but not in plastids . The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences . Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bombarded sample . The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by two homologous recombination events via the flanking ptDNA sequences . The transplastomes thus generated are stable, and are maternally transmitted to the seed progeny . The pPRV vector series targets insertions between the divergently transcribed trnV gene and the rps12/7 operon . The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for the study of transgene promoter activity.

J Mol Biol, 1994 Sep 23, 242(3), 302 - 5
Crystallization and preliminary X-ray analysis of an Escherichia coli purine repressor-hypoxanthine-DNA complex; Schumacher MA et al.; The purine repressor (PurR) is a DNA-binding protein, which together with a purine corepressor serves to regulate de novo purine and pyrimidine biosynthesis in Escherichia coli . PurR belongs to the structurally homologous lac repressor family of transcription regulators . A PurR-hypoxanthine-DNA complex has been crystallized, with DNA encompassing the high affinity purF operator site and which is 16 base-pairs long with 5'-deoxynucleoside overhangs on each complementary strand . The crystals diffract to better than 2.6 A and take the orthorhombic space group C222(1), with unit cell dimensions a = 175.9 A, b = 94.8 A and c = 81.8 A . The structure determination of this PurR-hypoxanthine-DNA complex will provide the first high resolution view of a Lacl member-DNA complex.

J Mol Biol, 1994 Sep 23, 242(3), 186 - 92
In vitro selection of small RNAs that bind to Escherichia coli phenylalanyl-tRNA synthetase; Peterson ET et al.; Small RNAs were selected from a highly degenerate library on the basis of their ability to bind tightly to Escherichia coli phenylalanyl-tRNA synthetase (FRS) . The 63 nucleotide library consisted of the acceptor stem and portions of the D and T stems of E . coli tRNA(Phe) flanking a 32 nucleotide randomized region . Because FRS binding relies on a correctly folded tRNA substrate, the selected variants from this library were expected to resemble tRNA(Phe) structure . After seven cycles of selection, the RNA library bound to FRS with similar affinity to that of the E . coli tRNA(Phe), but did not show detectable aminoacylation . Fourteen FRS-specific isolates were sequenced and found to contain an anticodon stem-loop including the anticodon triplet of tRNA(Phe) . The tight-binding RNAs fell into two classes depending on the location of this step-loop within the sequence . The acceptor stem defined by the non-randomized sequence was also found to be essential for binding . Mutation of two residues within a common hexanucleotide sequence present in one of the classes reduced binding to FRS . Taken together, these results suggest that in order to bind RNAs tightly, FRS requires the simultaneous interaction of the anticodon stem-loop and acceptor stem, and additional sequences needed for proper folding . This approach should assist in the detection of motifs that resemble tRNA, but are too dissimilar to be identified by sequence comparison.

J Biol Chem, 1994 Sep 23, 269(38), 23830 - 7
Human GMP synthetase . Protein purification, cloning, and functional expression of cDNA; Hirst M et al.; GMP synthetase is a key enzyme in the de novo synthesis of guanine nucleotides . Human GMP synthetase has been purified to homogeneity, and a cDNA encoding the enzyme has been isolated from the T-lymphoblastoma cell line, A3.01 . The open reading frame encodes a protein of 693 amino acids with a predicted molecular weight of 76,725 . The cDNA complements a guaA mutant of Escherichia coli, which lacks a functional GMP synthetase and extracts from the transformed E . coli exhibit GMP synthetase activity, which is absent in the parental strain . RNA hybridization analysis shows that human GMP synthetase is encoded by a single 2.4-kilobase message . DNA hybridization analysis suggests that the human GMP synthetase is encoded by one gene . In several human cell lines, the level of mRNA expression is substantially higher in proliferating, transformed cells than in nontransformed cells . In two transformed cell lines, treatment with phorbol ester inhibits proliferation and results in a dramatic down-regulation in the levels of GMP synthetase mRNA and protein.

J Biol Chem, 1994 Sep 23, 269(38), 23776 - 83
Identification of the nuclear and nucleolar localization signals of the protein p120 . Interaction with translocation protein B23; Valdez BC et al.; The human p120 nucleolar protein is a cell cycle-related protein that peaks during the S phase and has been shown to be associated with a beaded fibrillar structure . To study domains responsible for the nucleolar localization of protein p120, initially deletion mutants were made that defined sequences containing the localization signals; then, fusion genes that were composed of segments of the p120 molecule joined to the N-terminal end of the Escherichia coli beta-galactosidase were constructed . In the absence of the localization signals the beta-galactosidase remained in the cytoplasm . When the identified nuclear localization signal containing the amino acid sequence 99-110 (NAPRGKKRPAPG) was fused to the beta-galactosidase, the protein localized to the nucleus . When only the identified nucleolar localization signal containing the amino acid sequence 40-57 (SKRLSSRARKRAAKRRLG) was fused to the beta-galactosidase, the fusion protein remained in the cytoplasm . When both the nuclear and nucleolar localization signals were fused to the beta-galactosidase it localized predominantly to the nucleolus . Nucleolar protein B23, a putative "shuttle protein," bound to amino acid sequence 24-56 of protein p120 . Deletion analysis showed that amino acids 187-215 of protein B23 bound to protein p120 . The results suggest that protein B23 may be part of the mechanism of protein targeting to the nucleolus.

J Biol Chem, 1994 Sep 23, 269(38), 23625 - 31
Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY, SecE, and SecG (p12); Hanada M et al.; A cytoplasmic membrane protein, p12, of Escherichia coli was discovered as a new factor that stimulates the protein translocation activity reconstituted with SecA, SecY, and SecE (Nishiyama, K., Mizushima, S., and Tokuda, H . (1993) EMBO J . 12, 3409-3415) . Direct involvement of p12 in protein translocation was subsequently demonstrated in vivo by genetic studies, and the name SecG has been proposed for p12 (Nishiyama, K., Hanada, M., and Tokuda, H . (1994) EMBO J . 13, 3272-3277) . To elucidate the role of SecG in protein translocation and to characterize the translocation apparatus comprising these four Sec proteins, the activity of reconstituted proteoliposomes was examined in detail as a function of the amount of each component . SecG markedly stimulated the translocation activity over wide ranges of amounts of the other three Sec proteins, indicating that none of the other three Sec proteins substitutes for the SecG function . Detailed kinetic analyses indicated that the activity of proteoliposomes was dependent on the amount of the SecY-SecE complex when SecG was absent and the amount of the SecY.SecE.SecG complex when the proteoliposomes contained SecG . The translocation activity of the latter complex was significantly higher than that of the former one . Binding of SecA to liposomes appreciably increased when they contained both SecY and SecE, whereas the further presence of SecG did not enhance the binding . On the other hand, the ATPase activity of SecA, which was dependent on proOmpA and SecY.SecE-containing proteoliposomes, was significantly enhanced when the proteoliposomes contained SecG . Taken together, these results indicate that SecG enhances the translocation activity of the apparatus after the step of SecA targeting to SecY.SecE.

J Biol Chem, 1994 Sep 23, 269(38), 23477 - 83
Mode of membrane interaction of wild-type and mutant signal peptides of the Escherichia coli outer membrane protein A; Sankaram MB et al.; The membrane insertion potentials of the signal peptide of the outer membrane protein A (OmpA) from Escherichia coli and two peptides corresponding to functionally impaired mutant OmpA signal sequences were examined using spin label electron spin resonance (ESR) spectroscopy . The wild-type OmpA signal peptide, WT, a deletion mutant lacking the amino acid stretch 6-9, delta 6-9, and a substitution mutant with the isoleucine residue at position 8 replaced by asparagine, I8N, were incorporated into mixed lipid vesicles containing negatively charged 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) . Spin-labeled derivatives of phosphatidylglycerol and phosphatidylethanolamine containing a nitroxide moiety at the 12th position in the sn-2 acyl chain, 12-PGSL and 12-PESL, respectively, were employed for the ESR experiments . The 12-PGSL and 12-PESL exhibited two-component spectra in the presence of the WT and delta 6-9, but not when I8N was present . Using difference spectroscopy, the number of POPG and POPE molecules associated with an ordered lipid layer surrounding the peptides was estimated . The results suggest that WT exists as a transmembrane monomer in the membrane . The delta 6-9 mutant signal peptide appears to exist either as a transmembrane aggregate or partially inserted into the acyl chain region . The substitution mutant, I8N, has a most probable location near the membrane surface . Among these variants of the OmpA signal peptide, the ability to adopt a transmembrane monomeric orientation correlates well with the export activity.

What Is MIC?, What Is Growth Medium?, What Is Environmental Microbiology?, What Is Biofilter?, What Is Molecular Microbiology?, s, Microorganism, i, Bacteria, n, Bacterium, a, Microbiology, c, Bacteriology, a, Escherichia coli, a, Enterobacteriacea, a, Escherichia coli, s, Pseudomonas aeruginosa, o, Bacteria, s, Salmonella, s, Escherichia coli, c, Salmonella typhimurium, c, Escherichia coli, c, Microorganisms, s, Sepsis, i, Escherichia coli, r, Meningococcus, n, Antibiotics, e, Salmonella, c, Eutrophicated, o, Escherichia coli, r, Schizosaccharomyces, i, Cell suspensions, n, Bacillus, c, Gram positive

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

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

Last modified: May 25, 2005