Escherichia coli

J Biol Chem, 1996 Apr 12, 271(15), 8855 - 62
Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease . A study using oligodeoxynucleotide phosphorothioates; Thorogood H et al.; A set of phosphorothioate-containing oligonucleotides based on pGACGATATCGTC, a self-complementary dodecamer that contains the EcoRV recognition sequence (GATATC), has been prepared . The phosphorothioate group has been individually introduced at the central nine phosphate positions and the two diastereomers produced at each site separated and purified . The Km and Vmax values found for each of these modified DNA molecules with the EcoRV restriction endonuclease have been determined and compared with those seen for the unmodified all-phosphate-containing dodecamer . This has enabled an evaluation of the roles that both of the non-esterified oxygen atoms in the individual phosphates play in DNA binding and hydrolysis by the endonuclease . The results have also been compared with crystal structures of the EcoRV endonuclease, complexed with an oligodeoxynucleotide, to allow further definition of phosphate group function during substrate binding and turnover . For further study, see the related article "Probing the Indirect Readout of the Restriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Backbone" (Wenz, A., Jeltsch, A., and Pingoud, A . (1996) J . Biol . Chem . 271, 5565-5573).

J Biol Chem, 1996 Apr 12, 271(15), 8692 - 9
Bradyrhizobium japonicum porphobilinogen synthase uses two Mg(II) and monovalent cations; Petrovich RM et al.; Bradyrhizobium japonicum porphobilinogen synthase (B . japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M . R . (1995) J . Biol . Chem . 270, 19823-19827) . B . japonicum PBGS defines a new class of PBGS protein, type IV (classified by metal ion content), which utilizes a catalytic MgA present at a stoichiometry of 4/octamer, an allosteric MgC present at a stoichiometry of 8/octamer, and a monovalent metal ion, K+ . However, the divalent MgB or ZnB present in some other PBGS is not present in B . japonicum PBGS . Under optimal conditions, the Kd for MgA is <0.2 microM, and the Kd for MgC is about 40 microM . The response of B . japonicum PBGS activity to monovalent and divalent cations is mutually dependent and varies dramatically with pH . B . japonicum PBGS is also found to undergo a dynamic equilibrium between active multimeric species and inactive monomers under assay conditions, a kinetic characteristic not reported for other PBGSs . B . japonicum PBGS is the first PBGS that has been rigorously demonstrated to lack a catalytic ZnA . However, consistent with prior predictions, B . japonicum PBGS can bind Zn(II) (presumably as ZnA) at a stoichiometry of 4/octamer with a Kd of 200 microM; but this high concentration is outside a physiologically significant range.

J Biol Chem, 1996 Apr 12, 271(15), 8605 - 11
Structure-function relations of smooth muscle calponin . The critical role of serine 175; Tang DC et al.; Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin . Both properties are lost following phosphorylation (primarily at serine 175) by protein kinase C or calmodulin-dependent protein kinase II . To evaluate further the functional importance of serine 175, wild-type calponin and three site-specific mutants (S175A, S175D, and S175T) were expressed in Escherichia coli and compared with calponin purified from chicken gizzard smooth muscle in terms of actin binding, actomyosin MgATPase inhibition, and phosphorylation by protein kinase C and calmodulin-dependent protein kinase II . The affinities of skeletal muscle F-actin for wild-type and S175T calponins were similar to that for the tissue-purified protein (Kd = 0.8, 1.3, and 1.0 microM, respectively), whereas the affinities for S175A and S175D calponins were much lower (Kd = 26.8 and 44.2 microM, respectively) . Tissue-purified, wild-type, and S175T calponins displayed comparable inhibition of the smooth muscle actin-activated myosin MgATPase, whereas S175A and S175D calponins were much less effective . Phosphorylation confirmed serine 175 as the principal site of phosphorylation by both kinases . These results indicate that the hydroxyl side chain at position 175 of calponin plays a critical role in the binding of calponin to actin and inhibition of the cross-bridge cycling rate.

Nature, 1996 Apr 11, 380(6574), 548 - 50
Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2; Colas P et al.; A network of interacting proteins controls the activity of cyclin-dependent kinase 2 (Cdk2) (refs 1,2) and governs the entry of higher eukaryotic cells into S phase . Analysis of this and other genetic regulatory networks would be facilitated by intracellular reagents that recognize specific targets and inhibit specific network connections . We report here the expression of a combinatorial library of constrained 20-residue peptides displayed by the active-site loop of Escherichia coli thioredoxin, and the use of a two-hybrid system to select those that bind human Cdk2 . These peptide aptamers were designed to mimic the recognition function of the complementarity-determining regions of immunoglobulins . The aptamers recognized different epitopes on the Cdk2 surface with equilibrium dissociation constant in the nanomolar range; those tested inhibited Cdk2 activity . Our results show that peptide aptamers bear some analogies with monoclonal antibodies, with the advantages that they are isolated together with their coding genes, that their small size should allow their structures to be solved, and that they are designated to function inside cells.

Mol Gen Genet, 1996 Apr 10, 250(6), 705 - 14
Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli; Olsson CL et al.; To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI and rpIT genes encoding IF3, L35 and L20, respectively, were placed under the control of lac promotor/operator sequences . Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium . Lysogenic strains were constructed with recombinant lambda phages that express either rpmI and rplT or infC and prmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations . At low cellular concentration of IF3, but not L20, decreases and the growth rate slows . Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo . During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis . As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation of infC . The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein . In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41-43S is seen . Previous studies have shown that the L20 protein negatively controls its own gene expression . Reduction of the cellular concentration of L20 derepresses the expression of an rplT-lacZ gene fusion, thus confirming autogenous regulation by L20.

Mol Gen Genet, 1996 Apr 10, 250(6), 674 - 80
The isfA mutation inhibits mutator activity and processing of UmuD protein in Escherichia coli recA730 strains; Bebenek A et al.; Further studies on the isfA mutation responsible for anti-SOS and antimutagenic activities in Escherichia coli are described . We have previously shown that the isfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity . The isfA mutation has now been demonstrated also to suppress mutator activity in E . coli recA730 and recA730 lexA51(Def) strains that constitutively express RecA coprotease activity . We further show that the antimutator activity of the isfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD . Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in the recA730 isfA strain and partially restores its mutator activity . On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.

Biochemistry, 1996 Apr 9, 35(14), 4609 - 18
Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding; Thrall SH et al.; A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome . We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus . Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived . Titration of RT with Escherichia coli tRNA2Glu, E . coli tRNA2Tyr, E . coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys . The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT . Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys . The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding . The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA . The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites . Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands . The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA and p/t binding . Reverse transcriptase was found to bind to the mutant tRNA 10-fold more tightly than to the unaltered tRNA (Kd = 4.5 +/- 1.0 and 44.6 +/- 6.6 nM, respectively) . Further analyses revealed that the tighter affinity is probably due to a preferred p/t binding mode and not to one expected if separate tRNA and p/t binding regions are accessed simultaneously by the same molecule.

Biochemistry, 1996 Apr 9, 35(14), 4591 - 601
Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87; Annand RR et al.; In bee venom phospholipase A2, histidine-34 probably functions as a Bronsted base to deprotonate the attacking water . Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34 . We have prepared mutants at these positions and studied their kinetic properties . The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50-100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity . The interfacial Michaelis constants are changed by less than 10-fold for all mutants . Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis . The pKa of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4'-nitroacetophenone . The pKa is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pKa is unchanged . These pKas are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong . The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis.

Biochemistry, 1996 Apr 9, 35(14), 4468 - 79
Identification of active site residues of chorismate mutase-prephenate dehydrogenase from Escherichia coli; Christendat D et al.; Chemical modification studies of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase and mass spectral analysis of peptide fragments containing modified residues are presented . The reaction with diethyl pyrocarbonate (DEPC) results in the modification of several enzymic groups, including a single histidine group essential for dehydrogenase activity and a single lysine residue essential for mutase activity . This conclusion is based on the following evidence . (1) Hydroxylamine rapidly restores dehydrogenase activity to the DEPC-inactivated enzyme without restoring mutase activity . (2) Mutase activity is also lost upon treatment of the enzyme with trinitrobenzene sulfonate . (3) The reactivity of the dehydrogenase to DEPC increases with pH, suggesting the participation of a group with a pKa of 7.0 in the dehydrogenase reaction . (4) Two peptides identified by differential peptide mapping had mass values matching those calculated for peptides comprising residues 127-135 (containing His131) and residues 36-48 (containing Lys37) . In support of the idea that the residues being modified are within the active sites, we show that the substrates prephenate and nicotinamide adenine dinucleotide (NAD+) offer protection against inactivation of dehydrogenase activity while inactivation of mutase activity can be prevented by prephenate and a transition state analogue (3-endo-8-exo)-8-hydroxy-2-oxabicyclo{3.3.1}-non-6-ene-3,5-dicarboxylic acid (endo-oxabicyclic diacid) . Lys37 is conserved among several chorismate mutases and may participate in catalysis by interacting with an ether oxygen between C-5 and C-8 of chorismate in the transition state . His131 may be assisting in a hydride transfer from prephenate to NAD+ in the dehydrogenase reaction.

Biochemistry, 1996 Apr 9, 35(14), 4427 - 33
The role of the insertion loop around tryptophan 148 in tthe activity of thrombin; DiBella EE et al.; Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates . To probe the nature of the specificity of thrombin, a mutant has been constructed in which the Trp148 loop of thrombin has been replaced with the same loop of bovine trypsin . This mutant was expressed in Escherichia coli as prethrombin-2(148) using a T7 expression system previously described for wild-type prethrombin-2 {DiBella et al . (1995) J . Biol . Chem . 270, 163-169} . After refolding and purification, prethrombin-2(148) was activated to thrombin(148) with Echis carinatus snake venom . The k(cat)/K(m) for the release of fibrinopeptide A from fibrinogen was 4.5 +/- 0.5 microM(-1)s(-1) for thrombin(148), which was approximately 20% of that of recombinant thrombin (25 +/- 2.0 microM(-1)s(-1)) . Thrombin(148) was inhibited less well by hirudin with a K(i) of 500 pM compared to a value of 12 pM determined for recombinant thrombin . The mutant thrombin was also compared to trypsin and wild-type recombinant thrombin for the ability to cleave small peptide substrates . The Michaelis constants (K(m)) were found to be between 5- and 10-fold higher for thrombin(148) relative to wild-type recombinant thrombin, although the catalytic constants (k(cat)) for thrombin(148) and recombinant thrombin remained relatively unchanged for all three substrates . Thrombin(148) had a specificity constant (k(cat)/K(m)) 2-fold higher for the hydrolysis of H-D-phenyalanyl-L- pipecolyl-L-arginine-p-nitroaniline (a thrombin substrate) than that of trypsin . For N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine- p-nitroaniline (a trypsin substrate) and N-carbobenzoxyglycylprolyl-L-arginine-p-nitroaniline (a substrate for both enzymes), the specificity constants for trypsin were 1000- and 16-fold higher, respectively . Although replacement of the Trp(148) loop does not yield an enzyme with more trypsin-like specificity, the Trp(148) loop is important in the substrate binding and specificity of thrombin (on the basis of K(m) and K(i)).

Biochemistry, 1996 Apr 9, 35(14), 4359 - 64
His257 is a uniquely important histidine residue for tetracycline/H+ antiport function but not mandatory for full activity of the transposon Tn10-encoded metal-tetracycline/H+ antiporter; Yamaguchi A et al.; His257 is the only histidine residue located in the putative transmembrane region of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA) and contributes to the substrate/H+ coupling {Yamaguchi, A. . Adachi, K., Akasaka . T., Ono . N., & Sawai, T . (1991) J . Biol . Chem . 266, 6045-6051} . Tn10-TetA contains five histidine residues, including His257 . When these histidine residues were replaced by alanine one by one, only the His257Ala mutant showed almost complete loss of the tetracycline transport activity, whereas the other four His --> Ala mutants, H42A, H158A, H329A, and H359A, retained transport activity comparable to that of the wild type . The mutant which contains only one histidine, His257, retained about 80% of the wild-type activity, whereas the histidine-less mutant, in which all five histidine residues were replaced by Ala, exhibited little activity . These results clearly indicated that His257 is a unique histidine residue in TetA responsible for the transport activity . The His257Tyr mutant, irrespective of the presence or absence of the other four histidine residues, retained about 30% of the wild-type tetracycline transport activity and showed corresponding tetracycline-coupled H+ translocation, indicating that an imidazole group is not necessary at position 257 for the substrate/H+ coupling . A histidine-specific reagent, diethyl pyrocarbonate (DEPC), equally inactivated the wild-type and one-histidine mutant TetA, whereas the H257Y mutant was hardly inactivated by DEPC irrespective of the presence or absence of the other four histidine residues, indicating that the inactivation by DEPC is due to the modification of His257.

Biochemistry, 1996 Apr 9, 35(14), 4326 - 33
Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras; Pan CQ et al.; The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites . Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid . The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome . All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses . A newly discovered site resides in the lac operon of Escherichia coli . The binding of Fis to this site may antagonize repression by the LacI repressor . These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.

Biochemistry, 1996 Apr 9, 35(14), 4279 - 86
Structure of the LexA repressor-DNA complex probed by affinity cleavage and affinity photo-cross-linking; Dumoulin P et al.; The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage . These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator . In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein . In all four cases, the original amino acid was an arginine . The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as inferred from the structure of the LexA DNA binding domain {Fogh et al . (1994) EMBO J . 13, 3936-3944} . In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA . The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid . To reduce hydroxyl radical diffusion, the EDTA-iron cleavage reactions were done in the presence of high amounts of glycerol . The results indicate that amino acids 7 and 52 are near nucleotide pairs 8-12 of the operator and that amino acids 28 and 36 of LexA are near nucleotide pairs 5-8 of the operator . The results unambiguously define the orientation of the LexA DNA binding domain relative to the operator and provide support for the model of the LexA-operator complex proposed by Knegtel et al . {(1995) Proteins 21, 226-236} . Ethylation interference experiments further suggest that Arg-7 contacts the phosphate group between nucleotides 8 and 9 as predicted by the model.

J Comp Neurol, 1996 Apr 8, 367(3), 329 - 41
Interphotoreceptor retinoid-binding protein (IRBP): expression in the adult and developing Xenopus retina; Hessler RB et al.; Apposition of the neural retina and pigment epithelium is critical to photoreceptor development and function . Interphotoreceptor retinoid-binding protein (IRBP) is a major component of the extracellular matrix separating these epithelia in the African clawed frog Xenopus laevis (Gonzalez-Fernandez et al., {1993}, J . Cell Sci . 105:7-21) . In the adult retina, IRBP appears to mediate the transport of hydrophobic molecules, particularly retinoids and fatty acids, within the hydrophilic extracellular domain . In this paper, we compare the distribution of IRBP and its mRNA in adult and embryonic Xenopus retina . Xenopus IRBP antisense RNA, labeled with tritium or digoxigenin, was used for in situ hybridizaton studies . For immunohistochemistry, we used an antiserum against Xenopus IRBP expressed in Escherichia coli . In the adult, we found that IRBP is synthesized at similar levels by both rods and cones . The protein is restricted to the interphotoreceptor matrix, with lesser amounts in the pigment epithelial cytoplasm . In the embryo, expression of the mRNA for IRBP is restricted to the central retina, where photoreceptor differentiation has taken place . By contrast, the protein is distributed throughout the embryonic subretinal space . Therefore, the presence of IRBP precedes photoreceptor differentiation . In summary, IRBP is synthesized by both rods and cones and may be internalized by the pigment epithelium . In the embryo, IRBP is synthesized by the central retina and diffuses through the matrix, reaching the undifferentiated peripheral retina . In view of its ligand-binding properties, diffusion of IRBP may provide the peripheral neural retina with a vehicle to transport retinoids and docosahexaenoic acid (molecules critical to normal retinal development) from the pigment epithelium.

J Chromatogr A, 1996 Apr 5, 729(1-2), 113 - 24
Analysis of recombinant human interleukin-11 fusion protein derived from Escherichia coli lysate by combined size-exclusion and reversed-phase liquid chromatography; Amari JV et al.; A two-dimensional size-exclusion-reversed-phase high-performance liquid chromatographic assay has been developed for the quantitation of recombinant human interleukin-11 fusion protein (rhIL-11 FP) expressed in E . coli cells . The sample preparation procedure included the optimization of lysis buffer components to achieve maximum rhIL-11 FP recovery through the disruption of associations between rhIL-11 FP and E . coli components . The E . coli cells were dialyzed into lysis buffer and lysed by a French Press prior to two-dimensional chromatographic analysis . A size-exclusion column was used first to remove high- and low-molecular-mass E . coli components . Then reversed-phase chromatography was used to separate and quantify the rhIL-11 FP . The assay was linear over the range of 0.0294 to 0.235 mg/ml . The limit of quantitation, 0.0294 mg/ml, was based on % normalized residuals and precision criteria not exceeding 10% . The reproducibility of the assay for lysate samples was good on a daily (% R.S.D . = 1.0; n = 5) and a day-to-day reproducibility was good (% R.S.D . = 2.2; n = 9) . Selectivity and chromatographic peak identification were based upon gel electrophoresis and N-terminal sequencing of the rhIL-11 FP peak collected from the reversed-phase column.

J Mol Biol, 1996 Apr 5, 257(3), 700 - 15
Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase; Tibbitts TT et al.; In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51 . Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively . Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+ . Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme . At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme . Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme . The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased . Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme . To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2 . The two structures were then refined . The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51 . At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5 . In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone . Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.

J Mol Biol, 1996 Apr 5, 257(3), 574 - 85
Uneven distribution of GATC motifs in the Escherichia coli chromosome, its plasmids and its phages; Henaut A et al.; This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coli genome, compared to its distribution in phages and plasmids . At first sight the distribution of GATC words looks random . But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromasome), is used as a theoretical reference, strong biasesare observed . GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp . The last class is the only one present in E . coli parasites . It can be ascribed to the triggering sequences of the long-patch mismatch repair system . The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection . The other classes, which could be targets for a nucleic acid-binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs . Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions . In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions . In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.

J Mol Biol, 1996 Apr 5, 257(3), 550 - 60
Replication of plasmid R6K gamma origin in vivo and in vitro: dependence on IHF binding to the ihf1 site; Dellis S et al.; The gamma origin of plasmid R6K requires the specific initiator protein pi for initiation of replication . However, increased pi concentrations inhibit replication . The host-encoded integration host factor (IHF) protein permits gamma origin replication at otherwise inhibitory pi levels . IHF is thought to mediate this positive effect by directly binding to the gamma origin . In this study we demonstrate that IHF binding to one IHF site in the gama origin, ihf1, but not to the other side, ihf2, is necessary for the gamma origin to replicate at high pi protein levels . We also show that in vitro replication of the gamma origin plasmid requires IHF binding to the ihf1 site . Finally, we demonstrate both in vivo and in vitro that, when mutant pi proteins (hyperactive) are provided instead of wild-type pi, gamma origin plasmids can replicate in the absence of IHF . This supports a previously proposed hypothesis that the pi mutants can bypass the IHF requirement for gamma origin replication.

J Mol Biol, 1996 Apr 5, 257(3), 473 - 8
The side-chain of the amino acid residue in position 110 of the Lac repressor influences its allosteric equilibrium; Muller-Hartmann H et al.; Binding of the Lac repressor to its operator DNA controls the expression of the genes of the lac operon of Escherichia coli . Lac repressor's affinity for the lac operator is diminished by an inducer that affects the structure of the repressor tetramer . Here we report the cloning and sequencing of the mutant Lac repressor i-t gene, whose product, the LacR-t repressor, shows a higher affinity for the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and a lower affinity for the lac operator than the wild-type repressor . We show that the altered phenotype is due to a single amino acid residue replacement; the alanine residue at position 110 in the wild-type is replaced by threonine in i-t . Other amino acid residues in position 110 have been shown to result in an i-s phenotype . For the i-s-substitution of alanine 110 with lysine we demonstrate an increase in the affinity for operator DNA and a decrease in the affinity for IPTG . Thus, A110--> K shows the opposite effect to A110-->T on the repressor protein . We explain the phenotype of the LacR mutants by displacements of the conformational equilibrium for the dimeric repressor unit between RR (high operator affinity, low inducer affinity) and R*R* (low operator affinity, high inducer affinity) towards R*R* in the i-t and towards RR in the i-s mutant in position 110 with respect to the wild-type . The putative structures of the wild-type and mutant Lac repressors confirm this conclusion.

J Biol Chem, 1996 Apr 5, 271(14), 8502 - 8
The peripheral complex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G; Lepier A et al.; A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit . {14C}N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid . A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening . Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned . In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels . The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases . In contrast to the Vma10p protein, which behaved like a V0 subunit, the M . sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation . When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M . sexta V-ATPase could be purified by gel chromatography . This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase . We designate this new V1 component subunit G.

J Biol Chem, 1996 Apr 5, 271(14), 8152 - 6
Characterization of active recombinant his-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase; Hurtubise Y et al.; Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 . The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH . REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate . In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system . His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active . Its major features were compared to the untagged enzyme . In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a {2Fe-2S} Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+ . Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak . However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E . coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin.

J Biol Chem, 1996 Apr 5, 271(14), 8126 - 32
Dual regulation of a chimeric plant serine/threonine kinase by calcium and calcium/calmodulin; Takezawa D et al.; A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca2+-binding domain was recently cloned from plants (Patil, S., Takezawa, D., and Poovaiah, B . W . (1995) Proc . Natl . Acad . Sci . U . S . A . 92, 4797-4801) . The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca2+/calmodulin-dependent manner . The calmodulin-binding region of CCaMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII) . CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of 32P/mol) that is stimulated 3.4-fold by Ca2+ (0.339 mol of 32P/mol), while calmodulin inhibits Ca2+-stimulated autophosphorylation to the basal level . A deletion mutant lacking the visinin-like domain did not show Ca2+-stimulated autophosphorylation activity but retained Ca2+/calmodulin-dependent protein kinase activity at a reduced level . Ca2+-dependent mobility shift assays using E . coli-expressed protein from residues 358 520 revealed that Ca2+ binds to the visinin-like domain . Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca2+-induced conformational changes in the visinin-like domain . Autophosphorylation of CCaMK increases Ca2+/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its Ca2+-independent activity . This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca2+ and Ca2+/calmodulin . The presence of a visinin-like Ca2+-binding domain in CCaMK adds an additional Ca2+-sensing mechanism not previously known to exist in the Ca2+/calmodulin-mediated signaling cascade in plants.

J Biol Chem, 1996 Apr 5, 271(14), 8121 - 5
Elements within the first 17 amino acids of human osteonectin are responsible for binding to type V collagen; Xie RL et al.; The region in human osteonectin (ON) responsible for binding to type V collagen has been identified as the first 17 NH2-terminal residues . This conclusion is based upon binding studies with deletion mutants of ON produced in Escherichia coli, in which parts of the first 17 amino acids have been removed . Wild-type ON from E . coli and mammalian cell-derived nonglycosylated ON bind identically to type V collagen and at least twice as effectively as mammalian cell-derived N-glycosylated ON . In previous studies, it was shown that N-glycosylation at residue 99 significantly reduces the capacity of ON to bind to type V collagen . Results reported in this communication demonstrate that the actual binding site on ON for type V collagen is distal from the site of N-glycosylation in terms of amino acid sequence but may be proximal in the folded, fully glycosylated, three-dimensional structure . Consistent with this conclusion is the ability of a synthetic peptide consisting of amino acids 1-17 to specifically inhibit the binding of ON to type V collagen.

J Biol Chem, 1996 Apr 5, 271(14), 8095 - 100
Characterization of crystalline formate dehydrogenase H from Escherichia coli . Stabilization, EPR spectroscopy, and preliminary crystallographic analysis; Gladyshev VN et al.; The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex . The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme . FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable . The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined . Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400 . The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A . There is one monomer of FDH per crystallographic asymmetric unit . Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen . By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH . This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation . A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes . These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.

J Biol Chem, 1996 Apr 5, 271(14), 8046 - 52
Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies; Betton J et al.; The maltose-binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides . We have examined the characteristics of a Mal- mutant of malE corresponding to the double substitution Gly32 --> Asp/Ile33 --> Pro, MalE31, previously obtained by random mutagenesis . In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm . Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm . The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea . The renatured protein exhibits full maltose binding affinity (Kd= 9 x 10(-7) M), suggesting that its folded structure is similar to that of the wild-type protein . Unfolding/refolding experiments show that MalE31 is less stable (-5 . 5 kcal/mol) than the wild-type protein (-9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates . In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein.

J Biol Chem, 1996 Apr 5, 271(14), 8022 - 7
Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods . Cloning, heterologous expression, and localization; Frins S et al.; The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction . It is highly sensitive to the free calcium concentration ({Ca2+}) . The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein) . We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene . The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis . Recombinant GCAP was functionally expressed in Escherichia coli . Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free {Ca2+} with a Hill coefficient of 2.5 . Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free {Ca2+} . Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina.

J Biol Chem, 1996 Apr 5, 271(14), 8015 - 21
Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase; Zhang Y et al.; Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli . We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols . The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1 . The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme . Size exclusion chromatography and spectroscopic measurements at intermediate {GdnHCl} revealed two intermediate folding states . The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein . CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state . The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.

J Biol Chem, 1996 Apr 5, 271(14), 7978 - 85
Identification of novel Pax-2 binding sites by chromatin precipitation; Phelps DE et al.; The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues . The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation . In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned . Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells . In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain . The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2 . The genomic clones can now be used as sequence tags to identify potential target loci.

J Biol Chem, 1996 Apr 5, 271(14), 7923 - 6
Novel properties of L-type polypeptide subunits in mouse ferritin molecules; Beaumont C et al.; Properties of the L- and H-type polypeptide subunits forming ferritin 24-mer molecules in mice were investigated, using the products of in vitro transcription and translation from the two cloned genes, and recombinant ferritin molecules (H24L0 or H0L24) produced by transformation in Escherichia coli . Several different conditions for analytical electrophoresis reproducibly show that the relative migration position of the two mouse ferritin subunits is reversed from that reported for ferritin H- and L-subunits in all other mammals; since mouse and human H-polypeptides almost co-migrate, this unusual relative mobility is due largely to novel properties of the murine L-subunit . This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin . Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short-term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected . In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit . However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L-type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data.

Cell, 1996 Apr 5, 85(1), 71 - 81
Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation; Nishiyama K et al.; E . coli preprotein translocase comprises SecA and SecY/E/G complex . SecA delivers the preprotein to the putative protein-conducting channel formed by SecY/E by undergoing ATP-driven cycles of membrane insertion and deinsertion . SecG renders the translocase highly efficient . An antibody raised against the C-terminal region of SecG inhibits preprotein translocation into everted membrane vesicles despite the exposure of this region to the inside of membrane vesicles in the absence of preprotein translocation . When preprotein translocation was started with ATP and then blocked by the inhibition of ATP hydrolysis, the C-terminal region was exposed to the outside of membrane vesicles . Another region of SecG showed a change in membrane sidedness upon preprotein translocation, indicating that SecG undergoes topology inversion . This topology inversion was tightly coupled to the SecG function and linked with the insertion-deinsertion cycle of SecA.

Anal Biochem, 1996 Apr 5, 236(1), 101 - 6
Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose; McMahan SA et al.; Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose . This compound has a biotin as one functional group and a nitrilotriacetic acid as the other . The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites . The remaining two sites are available for binding to a histidine tag . The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence . Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit . This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose . The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot.

Eur J Pharmacol, 1996 Apr 4, 300(1-2), 99 - 104
Comparison of the effects of aminoguanidine and N omega-nitro-L-arginine methyl ester on the multiple organ dysfunction caused by endotoxaemia in the rat; Wu CC et al.; This study compares the effects of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide (NO) synthase, and N omega-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of endothelial NO synthase, on hypotension and multiple organ dysfunction caused by endotoxaemia in the anaesthetised rat . In the sham-operated rats, L-NAME, but not aminoguanidine, caused a dose-dependent increase in blood pressure . Endotoxin caused hypotension, increased in plasma nitrite (an indicator of inducible NO synthase activity), and dysfunction of kidney, liver and pancreas . Treatment of endotoxic rats with aminoguanidine or L-NAME caused significant and sustained rises in blood pressure . The increase in plasma nitrite caused by endotoxin was inhibited by aminoguanidine, but not by L-NAME . Aminoguanidine, but not L-NAME, attenuated the renal, liver and pancreatic dysfunction caused by endotoxaemia . Thus, selective inhibition of inducible (aminoguanidine), but not endothelial NO synthase (L-NAME) attenuates the circulatory failure and the multiple organ failure caused by endotoxaemia.

Nature, 1996 Apr 4, 380(6573), 451 - 3
Direct observation of single kinesin molecules moving along microtubules; Vale RD et al.; Kinesin is a two-headed motor protein that powers organelle transport along microtubules . Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule . Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead) . The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle . Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl . Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.

Biochim Biophys Acta, 1996 Apr 3, 1280(1), 41 - 50
Rapid transmembrane movement of C6-NBD-labeled phospholipids across the inner membrane of Escherichia coli; Huijbregts RP et al.; In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli . Exogenously added C6-NBD-labeled phospholipids rapidly flip across the inner membrane of E . coli, as was shown by a dithionite reduction assay applied to inverted inner membrane vesicles (IIMV) isolated from wild type E . coli cells . The rate of transmembrane movement of the phospholipid probes incorporated into IIMV is temperature dependent, and shows no phospholipid head group specificity . C6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t1/2 of 7 min . After the incorporation into IIMV C6-NBD-PG is partially converted to CL by CL-synthase . If IIMV are pretreated with proteinase K the conversion of this fluorescent probe to C6-NBD-CL is not observed anymore, suggesting that the catalytic domain of CL-synthase is at the cytoplasmic site of the plasma membrane of E . coli . Newly synthesized C6-NBD-CL also flips across the inner membrane although at a slower rate than the other phospholipid probes . The transmembrane movement occurs in both directions and is not influenced by treatment of the IIMV with a sulfhydryl reagent or a proteinase, nor by the presence of ATP, or a deltapH across the membrane of the IIMV . However, the transmembrane movement of the C6-NBD-labeled phospholipid probes is not observed in LUVETs (large unilamellar vesicles made by extrusion technique) prepared of wild type E . coli lipids, indicating that the rapid transmembrane movement of phospholipids across the inner membrane of E . coli is a protein-mediated process.

Biochemistry, 1996 Apr 2, 35(13), 4231 - 40
Roles of surface hydrophobic residues in the interfacial catalysis of bovine pancreatic phospholipase A2; Lee BI et al.; The interfacial binding is a unique and important step in the phospholipase A2 (PLA2) catalyzed hydrolysis of phospholipids which is distinct from the binding of a substrate to the active site . To assess the roles of surface hydrophobic residues of PLA2 in these processes, we selectively mutated Leu-19 and Leu-20 of bovine pancreatic PLA2 to charged (L19K and L20K), uncharged polar (L19S and L20S), and amphiphilic (L19W and L20W) groups and measured their kinetic and binding properties using various phospholipid aggregates, including micelles, monolayers, and polymerized mixed liposomes . The mutations of Leu-19 and Leu-20 did not significantly change either the tertiary structure or the thermodynamic stability of bovine pancreatic PLA2 . Toward monomeric 1,2-dihexanoyl-sn-glycero-3-phosphocholine, all Leu-20 mutants (L20S, L20W, and L20K) showed activities comparable to that of wild type whereas the substitution of Leu-19 with less hydrophobic side chains (L19S and L19K) reduced the activity to 70% and 50% . Toward zwitterionic 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC8PC) micelles, L20S and L20K mutants showed only 30% and 35% of the wild-type activity, respectively, whereas L20W was about twice as active as wild type . Also, L19S and L19K showed 75% and 15% of the wild-type activity, respectively . Toward anionic Trition X-100/sodium deoxycholate/diC8PC (4:2:1) mixed micelles, L20W and L20K were 2.6 times and twice more active than wild type . To determine the sn-2 acyl group selectivity of wild type and mutants, polymerized mixed liposomes were used which contained 1,2-bis{12-(lipoyloxy)-dodecanoyl}-sn-glycero-3-phosphoglycerol and 1 mol % of either 1-2{12-(1-pyrenebutanoyloxy)dodecanoyl}-2-hexanoyl-sn-glycero-3-++ +phosphocholine or 1-{12-(1-pyrenebutanoyloxy)dodecanoyl}-2-dodecanoyl-sn-glycero-3-+ ++phosphocholine . These measurements showed that Leu-19 was involved in the substrate binding and the sn-2 acyl group selectivity of bovine pancreatic PLA2 and that Leu-20 made a direct contact with the surface of phospholipid aggregates . The binding affinities of mutants to micelles, polymerized liposomes, and monolayers were well consistent with their kinetic behaviors, supporting the notion that the altered activities of Leu-19 mutants and Leu-20 mutants were due to the change in their substrate binding and interfacial binding, respectively . Finally, the L20W mutant represents the first example of protein engineering of PLA2 which results in a significant increase in interfacial binding to densely packed neutral monolayers and bilayers.

Biochemistry, 1996 Apr 2, 35(13), 4199 - 210
An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies; Marzabadi MR et al.; A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P) . A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme . The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site . A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding . This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding . Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM . As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate . Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date . However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P . The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite . Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site . However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P . Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite . As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site . Consequently, 4 is best described as an S3P-based substrate-analog inhibitor . These combined results corroborate the previous kinetic model {Gruys, K . J., Marzabadi, M . R., Pansegrau, P . D., & Sikorski, J . A . (1993) Arch . Biochem . Biophys . 304, 345-351}, which suggested that 4 interacts well with the S3P subsite but has little, if any, interaction at the expected glyphosate phosphonate or phosphoenolpyruvate-Pi subsites.

Biochemistry, 1996 Apr 2, 35(13), 4161 - 8
Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis; Pourcher T et al.; In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity . In general agreement with a previously suggested secondary structure model of melibiose permease {Botfield, M . C., Naguchi, K., Tsuchiya, T., & Wilson, T.H . (1992) J . Biol . Chem . 267, 1818}, clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence . Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops . Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane . Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it {Calamia, T., & Manoil, C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4937}, the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments . However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.

Biochemistry, 1996 Apr 2, 35(13), 4146 - 54
Mutation spectra of M13 vectors containing site-specific Cis-Syn, Trans-Syn-I, (6-4), and Dewar pyrimidone photoproducts of thymidylyl-(3'-->5')-thymidine in Escherichia coli under SOS conditions; Smith CA et al.; The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions . Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (-) strand synthesis with a DNA polymerase and dNTPs . Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F' cells (uvrA6 and phr-1) . The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (-) and (+) strands . Individual progeny of the photoproduct-containing (-) strands were plaque purified and sequenced by the dideoxy method . The cis-syn and trans-syn-I dimers were found not to be very mutagenic (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%) . The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector {Lawrence, C . W., et al . (1990) Mol . Gen Genet . 222, 166-168; LeClerc, J . E., et al . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 9685-9689} except that -1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3'-T-->C mutations was observed for the (6-4) photoproduct . Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side of the photoproduct . Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct . Analysis of the minor tandem mutations induced by the (6-4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability.

Biochemistry, 1996 Apr 2, 35(13), 4139 - 45
C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase; Glasfeld E et al.; Escherichia coli isoleucyl-tRNA synthetase is one of five closely related class I tRNA synthetases . The active site of the 939 amino acid polypeptide is in an N-terminal domain which contains an insertion believed essential for interactions with the tRNA acceptor helix . The enzyme was shown previously to contain an essential (for function in vivo) zinc bound to a Cys4 cluster at the C-terminal end of the polypeptide . The specific function of this zinc has been unknown . We show here that aminoacylation activity can be reconstituted in vitro by combining a 53 amino acid zinc-containing C-terminal peptide with a protein consisting of the remaining 886 amino acids . Reconstitution of aminoacylation is zinc-dependent . In contrast, the zinc-containing peptide is dispensable for synthesis of isoleucyl adenylate . Affinity coelectrophoresis showed that the 53 amino acid C-terminal peptide is required specifically for tRNA binding . We propose that the zinc-containing peptide curls back to the active site to make contact with the acceptor helix of bound tRNA, but not with isoleucine or ATP . It is the first example of a zinc-containing peptide in a class I tRNA synthetase that is essential for tRNA binding interactions . The design of this enzyme may be part of a more general scheme for class I tRNA synthetases to acquire acceptor helix binding elements during the development of the genetic code.

Biochemistry, 1996 Apr 2, 35(13), 4128 - 38
Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz{a,j}anthracene in M13mp7L2; Min Z et al.; The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz{a,j}-anthracene-3,4-diol 1,2-epoxide {(+)anti-DB{a,j}A-DE} that were site-specifically placed in a single-stranded M13mp7L2 replication vector . An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB{a,j}A-DE--trans-N2-dGuo or (+)anti-DB{a,j}A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation . In vitro studies using E . coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action . E . coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA . In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors . Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors . Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors . In JM103 cells, (+)anti-DB{a,j}A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB{a,j}A-DE--N2-dGuo induced exclusively G --> T transversions . In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct . While mutagenesis was SOS dependent in JM103 cells {<0.15% (-SOS) vs approximately 1.3% (+SOS)}, it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction) . It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms . In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.

Biochemistry, 1996 Apr 2, 35(13), 4079 - 83
Reversible oligomerization and denaturation of the chaperonin GroES; Seale JW et al.; The chaperonin GroEL can assist protein folding and normally acts with the co-chaperonin GroES . These Escherichia coli proteins are encoded on the same operon, with GroES positioned first . In this report, we have investigated the reversible folding of GroES . Using fluorescence anisotropy of dansyl-labeled GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity, and limited proteolysis, we show that GroES unfolds in a single, two-state transition . Importantly, intrinsic fluorescence and sedimentation velocity analyses show that GroES is capable of refolding and reassembling from a urea denatured state . The refolded GroES is fully active as shown by its ability to assist GroEL in the refolding of rhodanese . These results indicate that chaperonins may not require other chaperonins for successful folding/assembly . We also show that GroES is capable of assisting in the refolding/reassembly of fully denatured GroEL . The reversible folding of GroES coupled with the ability of GroES to assist the refolding/reassembly of GroEL suggest that the groE operon may be organized in a manner that provides a structural role in GroES/GroEL assembly as well as a functional role.

Biochemistry, 1996 Apr 2, 35(13), 4034 - 45
Equilibrium DNA binding of Sac7d protein from the hyperthermophile Sulfolobus acidocaldarius: fluorescence and circular dichroism studies; McAfee JG et al.; The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to double-stranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA . Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound . Reverse titration data have been fit directly to the McGhee-von Hippel model {McGhee, J., & von Hippel, P . (1974) J . Mol . Biol . 86, 469-489} using nonlinear regression . Sac7d binds noncooperatively to poly(dGdC) x poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KC1 (pH 6.8) . Some binding sequence preference is noted, with the binding to poly(dIdC) x poly(dIdC) over 10-fold stronger than to poly(DAdT) x poly(dAdT) . The binding is largely driven by the polyelectrolyte effect and is consistent with a release of 4.4 monovalent cations from DNA upon complex formation or the formation of 5 ion pairs at the protein-DNA interface . Extrapolation of salt back-titration data to 1 M KC1 indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy . A van't Hoff analysis of poly(dGdC) x poly(dGdC) binding shows that the binding enthalpy is approximately zero and the process is entropically driven . The affinity decreases slightly between pH 5.4 and 8.0 . There is no significant difference between the binding parameters of recombinant Sac7d and native Sac7 proteins, indicating that methylation of the native protein has no effect on the DNA binding function . The binding of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which shows a sigmoidal dependence on Sac7d concentration . The sigmoidal CD binding isotherm can be quantitatively modeled by a conformational transition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for poly(dIdC) x poly(dIdC) to 8 or less for poly(dAdG) x poly(dCdT).

Biochemistry, 1996 Apr 2, 35(13), 3950 - 6
Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification; Frillingos S et al.; By using site-directed chemical labeling of lactose permease, conformational changes induced by ligand binding are observed in the native membrane of Escherichia coli . Membranes containing permease mutants with a single-Cys residue and a biotin-acceptor domain were labeled with radioactive N-ethylmaleimide (NEM) in the presence or absence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or a proton electrochemical gradient, followed by solubilization in n-dodecyl beta-D-maltopyranoside and adsorption to avidin . TDG-induced enhancement of the reactivity of membrane-embedded Val315-->Cys (helix X) permease is observed, while the reactivity of Val331-->Cys (helix X) permease is inhibited by ligand binding or imposition of a proton electrochemical gradient . In contrast, the reactivity of permease with a single native Cys residue at position 148 (helix V) is blocked by TDG, but unaffected by the proton electrochemical gradient . Furthermore, as shown with right-side-out and inside-out membrane vesicles, the accessibility of Cys148 to either NEM or impermeant methanethiosulfonate derivatives is comparable from both sides of the membrane . On the other hand, TDG protects Cys148 from alkylation more effectively in right-side-out vesicles (apparent KD = 20-50 microM) than inside-out vesicles (apparent KD ca . 1.0 microM) . The findings provide strong support for the conclusion that the permease retains close to native conformation in n-dodecyl-beta-D-maltopyranoside . In addition, the results are consistent with the idea that lactose permease has two binding sites: one with higher affinity on the periplasmic surface of the membrane and another with lower affinity on the cytoplasmic surface.

Biochemistry, 1996 Apr 2, 35(13), 3933 - 43
Partitioning of HIV-1 Gag and Gag-related proteins to membranes; Ehrlich LS et al.; The binding of HIV-1 Gag and Gag-related proteins to model membranes was examined using three experimental systems: (i) large unilamellar phospholipid vesicles (LUVs) and recombinant Gag purified from Escherichia coli; (ii) LUVs added to a mammalian cell extract in which Gag proteins were expressed by a coupled transcription/translation system; and (iii) inside-out plasma membrane vesicles purified from human red blood cells (RBC) and recombinant, purified Gag from E . coli . Several novel aspects of HIV-1 Gag membrane interactions were observed: (i) Gag proteins bound with high affinity to both model membranes with a negatively charged surface and to RBC membranes . (ii) Binding of the Gag precursor and mature Gag proteins exhibited different sensitivities to ionic strength indicating that the precursor directed membrane binding through interactions that were qualitatively and quantitatively distinct from those of any of its individual domains . Studies using energy transfer between tryptophan residues in the proteins and anthroyloxy-containing probes inserted in the LUVs indicated that the orientation of the precursor and of the mature proteins on the membrane surface were distinct; (iii) Gag oligomers appear to have facilitated high-affinity binding under high salt conditions, suggesting that protein-protein interactions led to formation of stronger electrostatic or new hydrophobic membrane binding determinants . Since binding studies with model membranes permit quantitative analysis, these experimental approaches may permit identification of interactions that drive Gag assembly on the membrane.

Biochemistry, 1996 Apr 2, 35(13), 3925 - 32
Modulation of protein function by exogenous ligands in protein cavities: CO binding to a myoglobin cavity mutant containing unnatural proximal ligands; Decatur SM et al.; A variety of heterocyclic ligands can be exchanged into the proximal cavity of sperm whale myoglobin mutant H93G, providing a simple method for introduction of the equivalent of unnatural amino acid side chains into a functionally critical location in this protein . These modified proteins bind CO on the distal side . 1H NMR data on H93G(Im)CO, where Im is imidazole, demonstrate that the structure of the distal heme pocket in H93G(Im)CO is very similar to that of wild type; thus, the effects of the proximal ligand's properties on CO binding can be studied with minimal perturbation of distal pocket structure . The exogenous proximal ligands used in this study include imidazole (Im), 4-methylimidazole (4-MeIm), 4-bromoimidazole (4-BrIm), N-methylimidazole (N-MeIm), pyridine (Pyr), and 3-fluoropyridine (3-FPyr) . Substitution of the proximal ligand is found to produce substantial changes in the CO on and off rates, the equilibrium binding constant, and the vibrational stretch frequency of CO . Many of the changes are as large as those reported for distal pocket mutants prepared by site-directed mutagenesis . The ability to systematically vary the nature of the proximal ligand is exploited to test the effects of particular properties of the proximal ligand on CO binding . For example, 4-MeIm and 4-BrIm are similar in size and shape but differ significantly in pKa . The same relationship is true for Pyr and 3-FPyr . By comparison of the IR spectra and CO recombination kinetics of these complexes, the effects of proximal ligand pKa on the CO binding are assessed . Likewise, N-MeIm and 4-MeIm are similar in size and pKa but differ in their ability to hydrogen bond to amino acid residues in the proximal cavity . Comparisons of IR spectra and CO binding kinetics in these complexes reveal that proximal ligand conformation and hydrogen bonding affect the kinetics of CO binding . The mechanism of proximal ligand exchange between solution and the proximal cavity in CO complexes was investigated by obtaining the 19F NMR spectrum of H93G(3-FPyr)CO, whose 19F signal can be observed without interference from resonances of the protein . The proximal ligand is found to exchange within a few seconds by saturation transfer . This exchange rate is about 2 orders of magniture faster than what is observed for the isoelectronic metcyano complex {Decatur, S . M., & Boxer, S . G . (1995) Biochemistry 34, 2122-2129}; in both the ferrous CO and ferric cyano complexes, the proximal ligand exchange rate is independent of ligand concentration . These results suggest that the rate-limiting step in proximal ligand exchange is breakage of the iron-ligand bond, followed by rapid diffusion of the ligand through the protein to bulk solution.

Biochemistry, 1996 Apr 2, 35(13), 3880 - 5
Effects of protein RNase inhibitor and substrate on the quaternary structures of bovine seminal RNase; Murthy BS et al.; The effect of the protein RNase inhibitor (PRI) on the activity of bovine seminal RNase (BS-RNase) was investigated using the isolated quaternary forms, MxM and M=M, of the enzyme reported earlier {Piccoli, R., et al., (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 1870-1874} . We found that the inhibitor does not interact with the intact isolated forms but has dramatic, differential effects on the two forms when the assays are performed under reducing conditions . These conditions, which are essential for full activity of the inhibitor, and are typical of its cytosolic localization, also promote monomerization of the M=M form, while under identical conditions the MxM form becomes a noncovalent dimer (NCD) . The sensitivity of BS-RNase or that of the isolated quaternary forms under reducing conditions thus appears to be related to differential monomerization of the two forms of the enzyme; monomer being sensitive to PRI . The present study also shows that the interconversion between the two forms in equilibrium occurs at much higher rates in a reducing environment and that PRI further affects the interconversion and alters the equilibrium favoring monomerization of the protein . An opposite effect on the equilibrium between the forms is played by the substrate, which is found to stabilize the NCD form of the protein with a shift in the equilibrium between the two forms towards the dimer . These results are analyzed in the light of the antitumor action of the enzyme which is exerted in the cytosol, i.e., in the compartment housing the PRI and the ribosomal RNA, the molecular target of the enzyme.

Biochemistry, 1996 Apr 2, 35(13), 3875 - 9
Differentiation of catalytic sites on Escherichia coli F1ATPase by laser photoactivated labeling with {3H}-2-Azido-ATP using the mutant beta Glu381Cys:epsilonSer108Cys to identify different beta subunits by their interactions with gamma and epsilon subunits; Gruber G et al.; The ATP binding affinities of the catalytic sites in the three beta subunits of the Escherichia coli F1 ATPase (ECF1) have been explored in relation to the interaction of these subunits with the small subunits gamma and epsilon . ECF1 from the mutant beta E381C:epsilonS108C was reacted with different concentrations of {3H}-2-azido-ATP and covalent insertion of the nucleotide analogue induced by photoactivation of the azide group to a nitrene with single-pulse UV laser excitation . The enzyme showed cooperative binding of {3H}-2-azido-ATP in the presence of Mg2+ . The highest affinity site was located at betafree, the one of the three beta subunits in the mutant that does not form disulfide bonds with either the gamma or the epsilon subunit . This beta subunit is, therefore, the site of unisite catalysis in the enzyme . The second mole of {3H}-2-azido-ATP to bind was located in the beta subunit that links to epsilon (betaepsilon), while the lowest affinity binding of the substrate analogue was with the beta subunit that links to gamma (betagamma) . In the absence of Mg2+, all three beta subunits bound {3H}-2-azido-ATP with a similar, low affinity . The results show that binding of MgATP is determined by, and/or must determine, the interactions of the different alpha-beta subunit pairs with the single-copy subunits gamma, delta, and epsilon of the enzyme.

Mutat Res, 1996 Apr 2, 362(3), 261 - 8
The Escherichia coli DNA repair protein UvrA can re-associate with the UvrB: aflatoxin B1-DNA complex in vitro; Allan JM et al.; The UvrA and UvrB proteins form part of the UvrABc endonuclease, which is responsible for nucleotide excision repair in Escherichia coli . Using a mobility shift gel assay we have studied the binding of UvrA dimer, UvrB monomer and UvA(2)B trimer complexes with 40, 50 and 136 bp (32)P-end-labelled DNA fragments adducted with aflatoxin B(1) . UvrA was shown to re-associate with adduct specific UvrB: DNA complexes, a phenomenon which could be reversed by the addition of 500 mM potassium chloride or anti-UvrA anti-sera . Re-association was shown to be UvrA concentration dependent . Re-association of UvrA(2)B to the UvrB:DNA complex was not seen . We have also shown that the UvrB:DNA complex, in the case of aflatoxin B(1), is extremely stable with a half-life excess of 400 min and that fragment termini are not a specific substrate for UvrA binding.

Mutat Res, 1996 Apr 2, 362(3), 249 - 59
A comparison of the genotoxic effects of carboplatin and cisplatin in Escherichia coli; Overbeck TL et al.; cis-Diammine(1,1,-cyclobutanedicarboxylato)platinum(II) (carboplatin) is a second generation platinum anticancer agent with antineoplastic properties like that of its parent compound, cis-diamminedichloroplatinum(II) (cisplatin) but with substantially less deleterious side effects in treated patients with cisplatin . We compared their genotoxic effects in Escherichia coli and found carboplatin to be less cytotoxic (measured as loss of colony forming ability) that cisplatin in that equitoxic doses required greater than 60 time more carboplatin . However, solutions of carboplatin containing chloride ion became more cytotoxic to E . coli after a 24 h incubation period than similar freshly made solutions . Two platinum conversion products which were neither present in freshly made solutions nor in solutions lacking chloride were resolved by thin-layer chromatography (TLC) . One of the conversion products migrated like cisplatin and its occurrence in carboplatin solutions was associated with cisplatin-like properties, enhanced cytotoxicity and ability to induce the SOS responses in E . coli . The SOS-inducing abilities were determined by induction of a sulA::lacZ fusion . Likewise, adducts formed in end-labeled oligonucleotides treated with carboplatin appeared identical to those caused by cisplatin when carboplatin was preincubated in chloride-containing solutions but not by carboplatin in freshly made solutions . It is likely that responses evoked by carboplatin in biological systems are partly due to activation of carboplatin by its conversion of cisplatin.

Mutat Res, 1996 Apr 2, 362(3), 219 - 26
The ultraviolet-sensitizing function of plasmid R391 interferes with a late step of postreplication repair in Escherichia coli; Wang TC et al.; The conjugative plasmid R391 increases the UV radiation sensitivity of wild-type, uvrA, and lexA cells of Escherichia coli, but not recA strains . To investigate the UV-sensitizing function of R391, we examined the effect of R391 on the repair of DNA daughter-strand gaps and on the UV radiation sensitivities of various repair and/or recombination-deficient mutants . The presence of R391 did not significantly inhibit the repair of DNA daughter-strand gaps in uvrB cells . The presence of R391 increased the UV radiation sensitivity of uvrA, uvrA recF, uvrB, uvrB recF, uvrB recB, and uvrB ssb-113 cells to UV irradiation, but did not significantly increase the UV radiation sensitivity of uvrA ruvA and uvrA ruvC strains . Based on these results, we propose that the UV-sensitizing activity of R391 acts by inhibiting or interfering with the ruvABC-mediated postsynapsis step of recombinational repair.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 3007 - 10
The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets; Kato Y et al.; Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis . The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method . The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution . The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside . There is a 5-angstrom-diameter "hole" through the middle of the barrel . The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor . Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs . These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2969 - 74
Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide; Patel R et al.; Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site . During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template . The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide . The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored . The sequence requirements for template switching are compared to those for transposon excision.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2920 - 5
In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities; Sera T et al.; A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities . A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10 . These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3' . Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A) . Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions . The mutant proteins showed high specificity in a functional transcription interference assay . A model for the interaction of these mutants with the target DNA sequences is discussed.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2856 - 61
Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe; Fijalkowska IJ et al.; The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity . We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity . When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product) . When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable . However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E . coli mutL+ gene . These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe) . Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair . The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2801 - 6
Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse; Morgan BA et al.; Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA . The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use . For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids . In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity . In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice . Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively . With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription . Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation . Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2755 - 8
Protein synthesis editing by a DNA aptamer; Hale SP et al.; Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions . One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions . Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions . Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector . We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase . The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity . The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure . These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing . Thus, specific bases in a nucleic acid effector trigger the editing response.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2680 - 5
DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos; Walter J et al.; In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos . We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins . In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo . This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2670 - 4
The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle; Scherf U et al.; Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1 . CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis . Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells . Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS . CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell . The intracellular distribution of CAS resembles that of tubulin . In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle . CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells . CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles . Thus, CAS appears to be associated with but not to be an integral part of microtubules . Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon . CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum . These cells contain many microtubules . The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2652 - 7
Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites; Herzberg O et al.; The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart . The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase . Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain . While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers . This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate . The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.

Cell Stress Chaperones, 1996 Apr, 1(1), 78 - 89
Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer; Leung SM et al.; Differential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70) . Hsc70 had endothermic transitions with midpoints (Tm) at 59 degrees C and 63 degrees C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan . Combined with increased exposure at 60 degrees C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70 . An exothermic transition (Tm = 66 degrees C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes . An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c . A novel finding was an exothermic transition of Hsc70 beginning at about 30 degrees C (Tm = 41 degrees C) . No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range . Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation . However, Hsc70 was protease-resistant at 20 degrees C, sensitive at 40 degrees C and resistant when returned to 20 degrees C, indicating that this exotherm is associated with a reversible conformational change . As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c, a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK . For all of these substrates, the amount of complex formed increased with increasing temperature over the same range as the 41 degrees C exotherm . It is proposed that a conformational change exposes polar and charged residues in Hsc70 which subsequently become hydrated, resulting in an active chaperone . Hsc70 may be a thermal sensor that matches the supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells . Thermal activation of Hsc70 may also have a role in acquired thermotolerance.

Wei Sheng Wu Xue Bao, 1996 Apr, 36(2), 158 - 9
{Study on the death of Escherichia coli induced by hydrostatic pressures}; Qin L et al.; The effect of hydrostatic pressure on the death of E . coli was studied in this paper . The results indicated that E . coli could be killed by hydrostatic pressure above 800 bar . At 2300 bar E . coli was totally killed in 30 minutes . The time course of E . coli death induced by pressure indicated that the most E . coli was killed in the first 10 minutes after the pressure was applied . It was also found that the lower temperature favored killing E . coli under pressure.

Microb Drug Resist, 1996 Spring, 2(1), 155 - 7
Affinity chromatography as a means to study multienzyme complexes involved in murein synthesis; von Rechenberg M et al.; The interaction of murein hydrolases and synthases was studied by affinity chromatography . The lytic transglycosylases Slt70 and MltB of E . coli were purified and covalently linked to CNBr-activated Sepharose . Membrane extracts were analyzed for proteins that interact with the immobilized murein hydrolases . Slt70-Sepharose was found to retain the PBPs 1b, 1c, 2, and 3 . Likewise MltB-Sepharose enriched PBP 1b, 1c, and 3 . Thus both lytic transglycosylases have an affinity for a transpeptidase, PBP2 and/or 3, as well as for the bifunctional transpeptidase/transglycosylase 1b . Interestingly, in addition, the poorly characterized PBP 1c interacts strongly with both Slt70 and MltB . It is speculated that the lytic transglycosylases assemble a multienzyme complex consisting of hydrolases and synthases, which is involved in growth of the stress-bearing murein sacculus.

Microb Drug Resist, 1996 Spring, 2(1), 131 - 4
Inhibition of peptidoglycan hydrolase activity in vivo and in vitro by energy uncouplers in Escherichia coli; Rodionov DG et al.; The effects of energy uncouplers on in vivo and in vitro peptidoglycan hydrolase activities in Escherichia coli were determined . Sodium azide, potassium cyanide, and carbonyl cyanide m-chlorophenylhydrazone all inhibited ampicillin-induced lysis of exponential phase cultures, even when they were added to lysis-committed cultures . These energy uncouplers also inhibited the solubilization of radiolabeled peptidoglycan by bacterial suspensions that had been treated with 5% trichloroacetic acid by the method of Hartmann et al.3 to activate the peptidoglycan hydrolases . Therefore, the in vivo and in vitro activities of peptidoglycan hydrolases in E . coli are dependent on membrane energization.

Microb Drug Resist, 1996 Spring, 2(1), 99 - 103
Molecular interplay of murein synthases and murein hydrolases in Escherichia coli; Holtje JV; Affinity chromatography using different lytic transglycosylases as a specific ligand revealed an interaction of both murein hydrolases and murein synthases . This interaction is taken as evidence for the assemblage into a multienzyme complex that could function as a murein replicase precisely copying the given three-dimensional structure of the murein sacculus . The sacculus of the mother cell would function as a template, which is identically replicated by copying the lengths of the existing glycan strands and the pattern of crosslinkages . A hypothetical enzyme complex specifically involved in cell division and a complex specifically involved in cell elongation are presented . It is postulated that PBPs 1a and/or 1b are present in both complexes, whereas the presence of PBP2 or PBP3 defines the specificity of the murein-synthesizing machinery as being involved in either cell elongation or septation . Moreover, the proposed "holoenzyme" suprastructure could explain why the specific inhibition of PBPs 1a/1b results in bacteriolysis and why inhibition of PBP2 and PBP3 causes the well-known morphological alterations, spherical growth, and filamentation, respectively.

Microb Drug Resist, 1996 Spring, 2(1), 55 - 61
How does FtsZ find its location?
Voskuil JL, Nanninga N.
The conformational flexibility of FtsZ and the properties of its epitopes have been studied . Cellular fractions of Escherichia coli have been treated with Triton X-114 . FtsZ distributed in the polar as well as in the non-polar phase . This has been interpreted to mean that FtsZ can change its conformation . For the nonpolar conformation it has been assumed that the putative hydrophobic pocket of FtsZ (cf . Voskuil et al., J . Bacteriol . 176:1886-1893) is being turned inside out upon interaction with the cytoplasmic membrane . In a tentative model we suggest that FtsA mediates this interaction . Immunoprecipitations of FtsZ with various monoclonal antibodies in the presence or absence of 1 M NaCl gave a clue concerning the hydrophobicity and hydrophilicity of FtsZ's epitopes . Immunogold-labeling also showed differences with respect to the accessibility of FtsZ.

Microb Drug Resist, 1996 Spring, 2(1), 51 - 4
Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli; Vaganay S et al.; The D-glutamic acid-adding enzyme of Escherichia coli, or MurD, was purified from an overproducing strain and a few aspects of its reaction mechanism were studied . The existence of a reactive cysteinyl residue was shown by the following experiments: (1) two thiol-modifying reagents, (5,5'-dithiobis)2-nitrobenzoic acid and 2-nitro-5-thiocyanobenzoic acid, inactivated the enzyme; (2) incubation with tetranitromethane led to inactivation and to the appearance of cysteic acid (not to 3-nitrotyrosine); (3) in each case, ATP or UDP-MurNAc-L-Ala (but not D-glutamic acid) protected the enzyme from inactivation . The existence of a reactive lysyl residue was shown by the action of 2,4,6-trinitrobenzenesulfonic acid, a reagent specific for lysyl residues present in phosphate-binding sites . The formation of an acyl phosphate intermediate was consistent with three types of results: (1) the molecular isotope exchange reaction, which took place only in the presence of phosphate, but which was not strictly dependent on the presence of ADP; (2) a release of phosphate, measured by the molybdate assay, observed when the enzyme was incubated with ATP and UDP-MurNAc-L-Ala (without D-glutamic acid); (3) the appearance of a new radioactive compound (besides ATP and Pi) after incubation for a few minutes with UDP-MurNAc-L-Ala and {gamma-32P}ATP . Finally, the fact that phosphinate 1 was a good inhibitor of the enzyme (IC50 = 0.7 microM) strongly suggested that a tetrahedral transition state follows the acyl phosphate in the reaction pathway.

Microb Drug Resist, 1996 Spring, 2(1), 25 - 7
Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli; Liger D et al.; The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan . It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine . The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc . The murC gene product appears as a 50-kDa protein accounting for ca . 50% of total cell proteins . A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture . The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene . The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol . The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively . The specificity of the enzyme for its substrates was investigated with various analogues . Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.

J Mol Endocrinol, 1996 Apr, 16(2), 159 - 70
Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells; Nicholson LB et al.; We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease . Recombinant TSH-R.E used as immunogen, was produced in E . coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein . To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized . The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor . The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals) . Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells . The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule . None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG . The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function . The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.

Mol Chem Neuropathol, 1996 Apr, 27(3), 249 - 58
The in vitro formation of recombinant tau polymers: effect of phosphorylation and glycation; Ledesma MD et al.; Tau Isolated from paired helical filaments, aberrant structures that appear in Alzheimer disease (AD) patients' brains, show at least two posttranslational modifications: phosphorylation (Grundke-Iqbal et al., 1986; Ihara et al., 1986) and glycation (Ledesma et al., 1994; Yan et al., 1994) . To test whether these modifications could affect the capacity of tau to self-aggregate, recombinant tau was phosphorylated and glycated, and its capacity to form polymers analyzed . Our results indicate that on phosphorylation and glycation, the capacity of tau to form aggregates increases, and that glycation of tau could stabilize the assembled polymers and could facilitate formation of bundles from these polymers.

Biochem Mol Biol Int, 1996 Apr, 38(5), 957 - 64
Molcecular cloning of human antizyme cDNA; Yang D et al.; We have cloned the cDNA encoding the human ornithine decarboxylase antizyme from a 5'-stretch cDNA library of human B-cell lymphoma Daudi . The cloned human antizyme cDNA fragment consists of 1063 bp, has 80% homology to the rat antizyme cDNA, but shows almost no homology to the E . coli antizyme gene . Northern hybridization analysis shows that this gene is expressed in a number of human cell lines with an estimated mRNA transcript size of about 1.1 kb . The size of the mRNA suggests that the cloned cDNA fragment probably represents the full length of human antizyme mRNA transcript . Comparison of the human and rat antizymes demonstrates that they are highly conserved at both nucleotide and peptide levels.

Int J Biochem Cell Biol, 1996 Apr, 28(4), 451 - 6
Peptide degradation: effect of substrate phosphorylation on aminopeptidasic hydrolysis; Fernandez Murray P et al.; The effect of substrate phosphorylation on the susceptibility to exopeptidasic attack by leucyl aminopeptidase of swine kidney, alanyl aminopeptidase from human liver and aminopeptidase N of Escherichia coli was investigated using a synthetic heptapeptide (L-R-R-A-S-L-G) and its phosphorylated derivative . The enzyme-catalyzed products were analyzed by thin layer chromatography and electrophoresis . The sensitivities of peptide and phosphopeptide to leucyl aminopeptidase digestion were then compared . Data obtained indicated that when phosphopeptide was used as substrate one main product accumulated, which corresponded to the fragment A-S(P)-L-G, while unphosphorylated peptide was completely degraded to its constituent amino acids . Identical results were obtained using aminopeptidase N of E . coli . Using alanyl aminopeptidase as enzyme, the results obtained were essentially similar, since the exopeptidasic activity on the phosphorylated peptide was strongly hampered in the vicinity of phosphoseryl residue leading to accumulation of the same phosphorylated product, although this enzyme could not completely degrade the unphosphorylated peptide . It was concluded that phosphorylation of substrates does effect enzymic degradation of proteins.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 257 - 63
Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background; Schembri MA et al.; The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis . Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein . The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E . coli K-12 strain PC31 . Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors . Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene . These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 241 - 5
Adhesion of bovine enterotoxigenic Escherichia coli (ETEC) by type 1-like fimbriae; Catani CF et al.; Enterotoxigenic Escherichia coli (STa+) strains were isolated from adult bovine with diarrhea . These strains did not express any known ETEC-specific adhesins . Although hemagglutination with rat and sheep erythrocytes was observed in the presence of D-mannose (MRHA), these strains also showed mannose-sensitive hemagglutination (MSHA) with guinea-pig erythrocytes . Electron microscopic studies revealed the presence of fimbria-like structures (provisionally called "F43ms") on bacterial cells grown at 37 degrees C but not on cells grown at 18 degrees C . However, it was observed by SDS-PAGE that the J-1 strain (F43ms+) produces a protein similar to F1 fimbriae, and this strain hybridized with a DNA probe for F1 fimbriae . Immunogold-labelling techniques indicated that a rabbit anti-serum is specific for F43ms fimbrial structures, but not for Type 1 fimbriae . The immunofluorescence test carried out with semipurified F43ms on bovine brush borders suggests that the fimbria-like structures are responsible for the adhesion to bovine epithelial cells.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 227 - 31
Behavioral analysis of single cells of Myxococcus xanthus in response to prey cells of Escherichia coli; McBride MJ et al.; Myxococcus xanthus cells move over surfaces by gliding motility . The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M . xanthus cells . We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria (Escherichia coli) . Wild-type cells of M . xanthus did not respond to microcolonies of E . coli until they made physical contact . Cells which penetrated a colony remained in the colony until all of the prey cells were digested . Cells of frz mutants also penetrated E . coli microcolonies and digested some of the E . coli cells, but they invariably abandoned the microcolony leaving their food source behind . These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M . xanthus cells.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 169 - 74
Promoter analysis of the catalase-peroxidase gene (cpeA) from Rhodobacter capsulatus; Forkl H et al.; The expression of the Rhodobacter capsulatus catalase-peroxidase (cpeA) was studied by in-frame fusions of the upstream region of the cpeA gene to a promoter-less lacZ gene . The transcription of the cpeA gene is about 20-50-fold higher under aerobic-dark than under anaerobic-light conditions . The promoter was localized within a 69-bp upstream DNA region . The transcription start site, determined by primer extension, is 28 bases upstream from the initiation codon, confirming the postulated promoter localized by deletion analysis . Deletion of the part of the upstream region specifically responsible for oxygen regulation resulted in constitutive expression of the cpeA gene.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 153 - 8
An autonomously replicating plasmid transforms Botrytis cinerea to phleomycin resistance; Santos M et al.; A transformation system has been developed for the pathogen fungus Botrytis cinerea, based on the utilization of the wide host plasmid pUT737 that contains the Sh ble gene, conferring resistance to phleomycin . Transformed protoplasts were regenerated at 10-25 micrograms ml(-1) of phleomycin, at a frequency of 25-40 transformants per microgram of DNA, and they were resistant up to 50 micrograms ml(-1) . Southern hybridization using undigested and digested total DNA showed the presence of circular autonomously replicating plasmid pUT737 in the transformants . Reisolated plasmid from transformed fungus transformed E . coli and rescued plasmid was identified as PUT737 . Transformants were grown for four generations under non-selective conditions and replicative plasmids were still detected . Plasmids present in all transformants at this stage had been modified from native pUT737 and showed the same size and configuration indicating that selection through stabilizing plasmid forms has happened.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 147 - 52
Cooperative MetR binding in the Escherichia coli glyA control region; Lorenz E et al.; We determined the relative binding affinity of the MetR protein for wild-type and mutant MetR binding sites 1 and 2 in the Escherichia coli glyA control region . The results show that MetR binding site 1 has a higher affinity for the MetR protein than binding site 2 . In addition, the results suggest that binding of MetR to the glyA promoter is cooperative . Mutations that decrease the ability of MetR to bind to either site 1 or site 2 have no significant effect on MetR's ability to bend DNA.

Genome, 1996 Apr, 39(2), 404 - 9
Molecular and biochemical characterization of two nucleoside diphosphate kinase cDNA clones from Flaveria bidentis; Ananvoranich S et al.; Two nucleoside diphosphate kinase cDNA clones have been isolated from Flaveria bidentis by immunoscreening of an expression library with a polyclonal antibody raised against Flaveria chloraefolia flavonol 3-sulfotransferase (F3-ST) . The clones represent members of a small multigene family in this species . The nucleotide sequences of the two cDNA clones show a high degree of sequence similarity to other reported nucleoside diphosphate kinases (NDPKs), including the putative human tumor suppressor gene NM23 and the Drosophila regulatory gene . When these cDNA clones were expressed in Escherichia coli, their gene products exhibited NDPK enzymatic activity . The immunocross reaction of the clones with the antibody raised against the F3-ST suggests a common immuno-epitope and a similarity of a nucleotide binding site for the two proteins.

Pediatr Pol, 1996 Apr, 71(4), 343 - 6
{Diagnosis and management of acute haematogenous osteomyelitis in newborns and infants}; Gawrych E et al.; Many newborns in Poland still suffer from acute hematogenous osteomyelitis . The paper discusses the principles of early diagnosis and treatment.

Bioessays, 1996 Apr, 18(4), 325 - 32
Cooperative relaxation of supercoils and periodic transcriptional initiation within polymerase batteries; Guptasarma P; Transcription and DNA supercoiling are known to be linked by a cause-effect relationship that operates in both directions . It is proposed here that this two-way relationship may be exploited by the E . coli genome to facilitate constitutive transcription of supercoil-sensitive genes by polymerase batteries made up of uniformly spaces RNA polymerase elongation complexes . Specifically, it is argued that (1) polymerases transcribing DNA in tandem cooperate to relax each other's transcription-driven positive supercoils; and (2) negative supercoils driven upstream by elongation complexes tend to be 'harnessed' and used to cooperatively (and periodically) initiate fresh transcription from promoters . Harnessing of transcription-driven negative supercoils is thought to be achieved through the erection of protein barriers to the rotational upstream propagation of supercoils from transcription events . The possible relevance of such cooperation amongst polymerases to the activation of transcription by DNA-binding protein factors is emphasized . Some testable predictions are made and implications are discussed.

Bioessays, 1996 Apr, 18(4), 309 - 15
A case of convergent evolution of nucleic acid binding modules; Graumann P et al.; Divergent evolution can explain how many proteins containing structurally similar domains, which perform a variety of related functions, have evolved from a relatively small number of modules or protein domains . However, it cannot explain how protein domains with similar, but distinguishable, functions and similar, but distinguishable, structures have evolved . Examples of this are the RNA-binding protein containing the RNA-binding domain (RBD), and a newly established protein group, the cold-shock domain (CSD) protein family . Both protein domains contain conserved RNP motifs on similar single-stranded nucleic acid-binding surfaces . Apart from the RNP motifs, which have a similar function, the two families show little similarity in topology or amino acid sequence . This can be considered an interesting example of convergent evolution at the molecular level . Previously, a beta-sheet surface was found to interact with RNA in non-homologous proteins from yeast, phage and man, revealing that this mode of RNA binding may be a widely recurring theme.

Am J Physiol, 1996 Apr, 270(4 Pt 2), R749 - 54
Repeated infusions of TNF-alpha cause attenuation of the thermal response and influence LPS fever in guinea pigs; Goldbach JM et al.; In conscious, freely moving guinea pigs, tumor necrosis factor (TNF)-alpha and TNF-beta, infused into the aortic arch within a period of 45 min at a dosage of 5 micrograms/kg, induced different thermal responses . TNF-alpha evoked a biphasic elevation of abdominal temperature, both phases together lasting longer than 6 h . In response to infusions of TNF-beta, the first phase, lasting approximately 120 min, was the same as was observed in response to TNF-alpha, whereas the longer second phase of temperature increase was missing . When the infusion of TNF-alpha was repeated four times at intervals of 3 days, the second phase of the increase in abdominal temperature (120-360 min after start of infusion) tended to decrease in response to the third and was significantly attenuated in response to the fourth infusion of TNF-alpha . A control group of guinea pigs received four infusions of solvent (0.9% sterile pyrogen-free saline) . Another 3 days after the fourth infusion of TNF-alpha or solvent, all animals were injected with 20 micrograms/kg bacterial lipopolysaccharide (LPS from Escherichia coli; intramuscular injection) . In those guinea pigs having developed a reduced responsiveness to TNF-alpha, the first phase of LPS-induced fever was significantly suppressed, whereas the second phase tended to be enhanced, compared with animals having received four infusions of solvent . In conclusion, guinea pigs develop a reduced responsiveness to TNF-alpha after its repeated administration . In the state of lower reactivity to exogenous TNF-alpha, a reduced response of the first phase of LPS-induced fever (during which endogenous TNF-alpha is released) can be observed . This indicates that endogenous TNF-alpha may contribute to LPS-induced fever only in the initial phase of the febrile response of guinea pigs.

Am J Physiol, 1996 Apr, 270(4 Pt 2), R693 - 703
Endotoxin shock: thermoregulatory mechanisms; Romanovsky AA et al.; To clarify mechanisms of hypothermia in lipopolysaccharide (LPS) shock, four experiments were conducted in 72 chronically instrumented Wistar rats . They were intended to accomplish the following: experiment 1, determine the dose of intravenous Escherichia coli LPS that induces a body temperature (Tb) fall at a minimal mortality {the dose chosen (0.5 mg/kg) was then used in experiments 2-4}; experiment 2, identify the time course of the arterial blood pressure (BP) fall (shock) during the response to LPS; experiment 3, measure threshold Tb values for skin vasodilation and activation of metabolic heat production (M) during the LPS shock; and experiment 4, ascertain behavioral thermoregulation in LPS shock . For experiments 1-3, rats were kept in restrainers; ambient temperature (Ta) was 26 degrees C . In experiment 4, rats freely moved in a thermogradient (18-33 degrees C) . Variables monitored were colonic (Tc) and tail skin (Tsk) temperatures (experiment 1); BP (experiment 2); hypothalamic temperature (Thy), M (from oxygen consumption), and Tsk (experiment 3); and preferred Ta (Tpr) and abdominal temperature (experiment 4) . In experiment 1, LPS induced no Tc changes at 0 mg/kg, a biphasic fever (no mortality) at 0.05 mg/kg, a biphasic hypothermia (42% mortality) at 0.5 mg/kg, and a rapid fall of Tc (100% mortality) at 5 mg/kg . LPS-induced (0.5 mg/kg) hypotension (experiment 2) occurred simultaneously with the first hypothermic phase; both Tc and BP reached their nadirs (-0.8 +/- 0.1 degrees C and -34 +/- 12 mmHg) at approximately 1.5 h post-LPS . The major autonomic mechanism of the shock hypothermia was a shift in the threshold Thy for M from 37.9 +/- 0.3 to 36.0 +/- 0.3 degrees C (experiment 3; P < 0.05) . In experiment 4, rats selected Tpr below 25 degrees C (vs . 28-30 degrees C in control; P < 0.05) throughout the duration of the shock; their Tb dropped to 36.2 +/- 0.3 degrees C (P < 0.05) . In sum, the LPS shock-associated hypothermia involves a decrease in the threshold Tb for M, the resultant widening of the interthreshold zone, and cold-seeking behavior.

Microbiology, 1996 Apr, 142 ( Pt 4), 889 - 99
Organization of ribosomal RNA genes from the footrot pathogen Dichelobacter nodosus; La Fontaine S et al.; Southern hybridization analysis revealed that there were three rrn loci within the genome of Dichelobacter nodosus, the causative organism of ovine footrot . These loci (rrnA, rrnB and rrnC) were isolated on recombinant lambda clones, and comprised 16S, 23S and 5S rRNA genes closely linked in that order . Sequence and primer extension analysis revealed the presence of putative genes encoding tRNA(Ile) and tRNA(Ala) within the 16S-23S spacer region, as well as a number of potential regulatory features . These elements included a single promoter, which was mapped upstream of the 16S rRNA gene and which was similar to Escherichia coli consensus promoter sequences, an AT-rich upstream region, a GC-rich motif that may be involved in stringent control, leader and spacer antitermination sequences, sites for ribonuclease processing, and a putative factor-independent terminator sequence . Potential open reading frames (ORFS) were identified within the regions flanking the rrn loci, with identical copies of the 3' terminal ORF present downstream of each rRNA operon . Determination of the complete sequence of the 5S rRNA gene, and derivation of the 5S rRNA secondary structure, further substantiated the 16S rRNA-based placement of D . nodosus within the gamma division of the Proteobacteria.

Microbiology, 1996 Apr, 142 ( Pt 4), 755 - 63
The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity in vivo by oxygen inhibition; D'mello R et al.; Cytochrome bd is a respiratory oxidase in Escherichia coli and many other bacteria . It contains cytochromes b558, b595 and d as redox centres, and is thus unrelated to the haem-copper super-family of terminal oxidases . The apparent affinities (Km) for oxygen uptake by respiring cells and membranes from a mutant lacking the alternative oxidase cytochrome bo' were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration . Respiration rates were maximal at oxygen concentrations of 25-50 nM, but the kinetics were complex and indicative of substrate (i.e . oxygen) inhibition . Km values were in the range 3-8 nM (the lowest recorded for a respiratory oxidase), and Ki values between 0.5 and 1.8 microM were obtained . Low temperature photodissociation of anoxic, CO-ligated membranes confirmed the absence of cytochrome bo' and revealed a high-spin b-type cytochrome identified as cytochrome b595 of the cytochrome bd complex . Photodissociation in the presence of oxygen revealed binding of a ligand (presumably oxygen) to cytochrome b595 at a rate much greater than that of CO binding, and formation of the oxygenated form of cytochrome d . The results confirm that both high-spin haems in the cytochrome bd complex bind CO and demonstrate that oxygen can also react with both haems . Substrate inhibition of oxidase activity, in addition to transcriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytochrome bd- and bo'-terminated respiratory pathways.

Plant Physiol, 1996 Apr, 110(4), 1197 - 205
Characterization of soybean choline kinase cDNAs and their expression in yeast and Escherichia coli; Monks DE et al.; An expressed sequence tag from Arabidopsis that displayed sequence homology to mammalian and yeast choline kinases was used to isolate choline kinase-like cDNAs from soybean (Glycine max L.) . Two distinct cDNAs, designated GmCK1 and GmCK2, were recovered that possessed full-length reading frames, each sharing approximately 32% identity at the predicted amino acid level with the rat choline kinase . A third unique choline kinase-like cDNA, GmCK3, was also identified but was not full length . Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and GmCK2 in both yeast and Escherichia coli demonstrated that each encodes choline kinase activity . In addition to choline, other potential substrates for the choline kinase enzyme include ethanolamine, monomethylethanolamine (MME), and dimethylethanolamine (DME) . Both soybean choline kinase isoforms demonstrated negligible ethanolamine kinase activity . Competitive inhibition assays, however, revealed very distinct differences in their responses to DME and MME . DME effectively inhibited only the GmCK2-encoded choline kinase activity . Although MME failed to effectively inhibit either reaction, an unexpected enhancement of choline kinase activity was observed specifically with the GmCK1-encoded enzyme . These results show that choline kinase is encoded by a small, multigene family in soybean comprising two or more distinct isoforms that exhibit both similarities and differences with regard to substrate specificity.

Proc Natl Sci Counc Repub China B, 1996 Apr, 20(2), 27 - 30
Single-step hybridization screening for recombinant DNA clones with correct insert orientation and intact junction using a junctional oligonucleotide probe; Au LC et al.; Cloning of DNA fragments with blunt ends or identical protruding ends results in colonies with the insertion sequence existing in two possible orientations . The orientation and the junctional sequence of the positive clones need to be established by means of restriction analysis and/or sequencing . Here, we proposed a rapid one-step method for the screening of clones not only with the desired orientation, but also with an intact junctional sequence . In this method, a 16-18 meric oligonucleotide probe synthesized according to the expected vector-insert junctional sequence is used as a probe in colony hybridization screening . Using this strategy, only recombinant clones which fulfill the above criteria will show positive signals in hybridization.

Am J Physiol, 1996 Apr, 270(4 Pt 1), G667 - 75
Effects of short-term endotoxemia and dopamine on mucosal oxygenation in porcine jejunum; Hasibeder W et al.; Effects of Escherichia coli lipopolysaccharide (2 micrograms.kg-1.20 min-1; LPS), given systemically (S) or via superior mesenteric artery (M), and consecutive dopamine infusion (16 micrograms.kg-1.20 min-1) on jejunal mucosal tissue O2 tension (PO2muc) and serosal tissue O2 tension (PO2ser; Clark-type surface electrodes) and jejunal mucosal microvascular hemoglobin O2 saturation (HbO2muc; tissue reflectance spectrophotometry) were investigated in a hemodynamically stable pig model . Twenty-one pigs were anesthetized, paralyzed, and mechanically ventilated . After laparotomy, a mesenteric venous catheter was inserted and a jejunal antimesenteric enterotomy performed . LPS-infused animals developed similar degrees of pulmonary hypertension . No differences in cardiac output and mean arterial blood pressure between groups were found . PO2muc and HbO2muc were significantly lower in M animals compared with control (C) {210 min; PO2muc: 7.12 +/- 1.81 (M), 19.01 +/- 3.12 mmHg (C); HbO2muc: 28.78 +/- 3.36 (M), 49.09 +/- 3.84% (C)}, whereas S animals ranged in between (PO2muc: 13.36 +/- 2.2 mmHg; HbO2muc: 40.68 +/- 4.43%) . Of measured PO2muc values, 12.6 (C), 20.6 (S), and 46.3% (M) ranged from 0 to 5 mmHg . PO2ser was lower in LPS animals compared with control {59.43 +/- 5.4 (C), 45.00 +/- 6.12 (S), 47.33 +/- 4.34 (M) mmHg} . Dopamine increased PO2muc and HbO2muc to similar absolute values and significantly decreased frequency of PO2muc (0-5 mmHg) in M animals . We conclude that LPS impairs mucosal tissue oxygenation independently of systemic hemodynamics . Mucosal microvascular dysfunction depends on regional LPS concentrations . Under conditions of compromised tissue oxygenation, dopamine significantly improves PO2muc and HbO2muc.

Am J Physiol, 1996 Apr, 270(4 Pt 1), G660 - 6
Endotoxin stimulates gene expression of ROS-eliminating pathways in rat hepatic endothelial and Kupffer cells; Spolarics Z; Reactive oxygen species (ROS) are mediators of cellular injury and play a putative role in the onset of hepatic damage during endotoxemia or sepsis . It has been suggested that induction of glucose-6-phosphate (G-6-P) dehydrogenase, the key enzyme of the hexose monophosphate shunt (HMS), may support ROS-producing or ROS-eliminating pathways in hepatic endothelial and Kupffer cells during endotoxemia . The aim of the study was to assess in vivo lipopolysaccharide (LPS)-induced alterations in rat gene expression of selected enzymes that are in functional relationship with the HMS . mRNA levels and activities of glucose transporter GLUT-1, Mn- and CuZn-dependent superoxide dismutases (Mn-SOD and CuZn-SOD), and Se-dependent glutathione peroxidase (Se-GPX) were determined . Cellular extracts were analyzed 7 or 22 h after injection of LPS (Escherichia coli, 2 mg/kg ip) or injection of saline . Exposure to LPS for 7 or 22 h caused a 10- to 25-fold increase in GLUT-1 mRNA levels in endothelial and Kupffer cells . In parenchymal cells, GLUT-1 mRNA expression was low, and LPS caused no marked changes . Cellular levels of Mn-SOD mRNA were 20-40 times greater in all hepatic cells from LPS-treated animals than in cells from control rats . LPS at 22 h increased Mn-SOD activity by 45% in endothelial cells but caused no significant changes in Kupffer or parenchymal cells . Message levels and enzyme activities of CuZn-SOD and Se-GPX were significantly elevated 22 h after LPS injection in endothelial cells only . Thus LPS results in marked upregulation of functionally related genes in hepatic cells . In endothelial cells, the simultaneous upregulation of GLUT-1, G-6-P dehydrogenase, Mn-SOD, CuZn-SOD, and Se-GPX may represent an important mechanism for accelerated elimination of ROS released from activated sinusoidal phagocytes . In Kupffer cells, upregulated GLUT-1 and G-6-P dehydrogenase, together with constitutively present SOD and lack of upregulated Se-GPX, suggest an elevated capacity to produce O2- and H2O2 that is consistent with primed bacterial killing.

Am J Physiol, 1996 Apr, 270(4 Pt 1), G634 - 45
Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions; Philpott DJ et al.; The effect of enteropathogenic Escherichia coli (EPEC) infection on electrophysiology of T84 cell monolayers was examined . After 18 h of infection with EPEC (E2348), transepithelial electrical resistance was decreased (30 +/- 5% of uninfected values) compared with monolayers infected with a nonpathogenic E . coli strain (104 +/- 13%) . Resistance of monolayers infected with EPEC mutant strain CVD206, deficient in attaching and effacing lesion formation, was partially reduced (66 +/- 10%) . In addition, permeability of EPEC-infected T84 monolayers increased compared with uninfected cells . Associated with these changes was an altered distribution of the tight junction protein, ZO-1 . Taken together, these findings suggest that the barrier defect induced by EPEC was at the level of the tight junction . Adenosine 3'5'-cyclic monophosphate-stimulated chloride secretion was also diminished in EPEC-infected cells, whereas Ca2+ -dependent chloride secretion was not different from uninfected cells . These findings indicate that EPEC infection alters intestinal epithelial barrier and transport functions . Furthermore, these results provide a possible mechanism for EPEC-induced diarrheal disease.

Am J Physiol, 1996 Apr, 270(4 Pt 1), E580 - 8
Hyperglucagonemia and hepatic glucose metabolism during infection in the conscious dog; McGuinness OP et al.; The chronic and acute roles of hyperglucagonemia in sustaining the increased glucose production observed in the conscious infected dog were examined . Three groups of dogs were studied: a sham group (SHAM; n = 10), an infected group (INFXN; n = 11), and a sham group in which the chronic (42-h) increase in glucagon observed in INFXN was simulated (SimGGN; n = 5) . INFXN and SimGGN were studied in the presence of hyperglucagonemia . In addition, glucagon was selectively decreased for 180 min in INFXN by use of somatostatin with basal intraportal insulin replacement and in SimGGN by discontinuing the exogenous glucagon infusion . Tracer and arteriovenous difference techniques were used to assess hepatic glucose metabolism and gluconeogenesis . Whereas the rate of glucose appearance (Ra) was increased by 30% (3.3 +/- 0.1 vs . 2.5 +/- 0.1 mg.kg-1.min-1) in INFXN vs . SHAM, Ra did not increase in SimGGN (2.4 +/- 0.2 mg.kg-1.min-1) . In addition, the 30% increase in net hepatic gluconeogenic precursor uptake seen in INFXN did not occur in SimGGN despite an augmented net hepatic alanine fractional extraction (0.62 +/- 0.03 vs . 0.47 +/- 0.05, SimGGN vs . INFXN) . With acute removal of hyperglucagonemia, endogenous Ra decreased in SimGGN and INFXN by 1.0 +/- 0.2 and 1.4 +/- 0.3 mg.kg-1.min-1, respectively . Net hepatic alanine fractional extraction in INFXN, leading to a greater rise in arterial blood alanine levels . In summary, chronic hyperglucagonemia alone cannot explain the increase in Ra observed during an infection . The marked hyperglucagonemia seen during infection plays an essential role in sustaining normal net hepatic fractional alanine extraction to compensate for an impairment in glucagon-stimulated hepatic amino acid transport activation.

Pflugers Arch, 1996 Apr, 431(6), 853 - 62
Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle; Strauss JD et al.; Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles . These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation . Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5 . For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis . Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC . Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 "mu"M), while Ca2+ sensitivity, i.e . the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution . At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 "mu"M and 150 "mu"M, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx . 0.2 units) . Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid . We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-) . The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers . Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI . However, each of the recombinant TnIs could substitute for endogenous TnI . In combination with exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution . The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

Mol Gen Mikrobiol Virusol, 1996 Apr-Jun, (2), 18 - 22
{Ability of a recombinant protein containing fragment 114-122 of myelin basic protein, to cause allergic encephalomyelitis in guinea pigs}; Kuvshinov VN et al.; Four genetic constructions have been designed, capable of producing in E . coli the hybrid beta-galactosidases containing the encephalitogenic determinant 114-122 of myelin basic protein . The ability of chromatography-purified proteins to cause allergic encephalomyelitis in guinea pigs has been investigated . Only one out of four proteins carrying at least one complete replica of encephalitogenic determinant did induce allergic encephalomyelitis in animals . Effects of the structural context of the encephalitogenic determinant on its functional activity are discussed.

Comp Biochem Physiol B Biochem Mol Biol, 1996 Apr, 113(4), 809 - 16
Expression of horseshoe crab arginine kinase in Escherichia coli and site-directed mutations of the reactive cysteine peptide; Strong SJ et al.; Arginine kinase (AK) from the horseshoe crab Limulus polyphemus was expressed in Escherichia coli . The bulk of expressed protein resided in insoluble inclusion bodies . However, approximately 3 mg enzyme protein/l culture was present as active soluble AK . The AK-containing expression vector construct was subjected to site-directed mutagenesis via a polymerase chain reaction-based megaprimer protocol . The AK reactive cysteine peptide was engineered so that it was identical to the corresponding peptide sequence of creatine kinase, another member of the guanidino kinase enzyme family . The resulting expressed protein had a considerably reduced specific activity but was still specific for arginine/arginine phosphate . No catalytic activity was observed with other guanidine substrates (creatine, glycocyamine, taurocyamine, lombricine) . The reactive cysteine peptide, characteristic of all guanidino kinases, very likely plays a minimal role in determining guanidine specificity.

Appl Environ Microbiol, 1996 Apr, 62(4), 1444 - 7
Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA; Vasina JA et al.; A transcriptional gene fusion between the cspA promoter and the lacZ gene was constructed to assess the usefulness of cold shock promoters for low-temperature protein expression . Synthesis of beta-galactosidase was efficiently repressed at 37 degrees C but rapidly induced upon transfer to the 15-to-30 degrees C range, leading to a three- to fivefold increase in specific activity relative to control cultures . Although the initial rates of beta-galactosidase accumulation at 20 degrees C were twice those measured at 15 degrees C, prolonged incubation at 20 degrees C, but not 15 degrees C, led to a dilution of activity due to repression of the promoter and cell division.

Appl Environ Microbiol, 1996 Apr, 62(4), 1416 - 23
Development and field application of a quantitative method for examining natural assemblages of protists with oligonucleotide probes; Lim EL et al.; A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists in cultures and environmental water samples is described . Filtration, hybridization, and enumeration of fixed cells with biotinylated eukaryote-specific probes and fluorescein isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, Mass.) . Counts of various species of cultured protists by this probe hybridization method were not significantly different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods . However, counts of total nanoplankton (TNAN) based on probe hybridizations in several field samples and in samples collected from a mesocosm experiment were frequently higher than TNAN counts obtained by staining with DAPI or AO . On the basis of these results, 25 to 70% of the TNAN determined with probes were not detectable by DAPI or AO staining . The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed to the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with distinguishing DAPI- or AO-stained protists attached to or embedded in aggregates . We conclude from samples examined in this study that enumeration of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains . The quantitative in situ hybridization method we have described here enables the direct enumeration of free-living protists in water samples with oligonucleotide probes . When combined with species-specific probes, this method will enable quantitative studies of the abundance and distribution of specific protistan taxa.

Int J Immunopharmacol, 1996 Apr, 18(4), 259 - 62
OM-85 BV upregulates the expression of adhesion molecules on phagocytes through a CD 14-independent pathway; Marchant A et al.; OM-85 BV is a preparation of bacterial extracts which proved to be of some efficacy in the prevention of respiratory tract infections . However, the mechanisms of action of this drug remain unclear . As we recently observed that OM-85 BV upregulates the expression of adhesion molecules on phagocytes, we took advantage of this property to determine whether the activating effects of OM-85 BV on monocytes and granulocytes depend on its interaction with CD14 molecules . Indeed, CD14 represents the major cell surface receptor for lipopolysaccharide and other bacterial products at the surface of leucocytes . First, we found that the upregulation of Mac-1 (CD11b/CD18) induced in vitro by OM-85 BV on monocytes was not blocked by an anti-CD14 monoclonal antibody (mAb) which inhibits monocyte responses to lipopolysaccharide . Similarly, the anti-CD14 mAb inhibited upregulation of Mac-1 on granulocytes when it was induced by lipopolysaccharide but not by OM-85 BV . To confirm that the effects of OM-85 on the expression of Mac-1 is CD14-independent, we analysed the responses of a patient with paroxysmal nocturnal haemoglobinuria, a disease associated with a defect of CD14 expression at the membrane of phagocytes . We found that monocytes and granulocytes of this patient displayed an impairment in Mac-1 upregulation in response to lipopolysaccharide whereas they responded normally to OM-85 BV . We conclude that OM-85 BV activates phagocytes through a CD14-independent pathway . The characterisation of the cell surface receptors of monocytes and granulocytes involved in the interactions with OM-85 BV might provide a molecular clue to the mode of action of this preparation of bacterial extracts.

Acta Virol, 1996 Apr, 40(2), 67 - 72
Immunodetection of beet necrotic yellow vein virus RNA3-encoded protein in different host plants and tissues; Li Y et al.; The protein p25 open reading frame (ORF) of beet necrotic yellow vein virus-BNYVV RNA3 was cloned into bacterial expression vector downstream of the 5-'terminus part of beta-galactosidase ORF and the expressed p25 fusion protein was used to produce an antiserum . The latter was employed to detect the subcellular location of p25 in mechanically inoculated Tetragonia expansa, Chenopodium quinoa and sugarbeet leaves by Western blot assay . The results showed that p25 was present as a soluble protein only in the S30 fraction of T . expansa, C . quinoa and sugarbeet leaves infected with BNYVV.

Biopolymers, 1996 Apr, 38(4), 471 - 91
Isolation, characterization, and magnesium-induced self-association kinetics of discrete aggregates of RecA protein from Escherichia coli; Budzynski DM et al.; Dynamic and static intensity light scattering techniques were employed to identify conditions allowing preparation of homogeneous solutions of distinct oligomeric states of RecA protein . These hydrodynamically distinguishable oligomer populations of RecA protein were obtained in homogeneous pure quantities sufficient for physical studies . Results indicate two fairly narrow distributions of RecA oligomers comprised on average of 42 +/- 3 and 18 +/- 1 RecA monomers . These structures, denoted RecA42 and RecA18, respectively, could be obtained reproducibly in milligram quantities and were stable for at least one week . This enabled reliable characterizations of their hydrodynamic properties by dynamic and total intensity light scattering . These measurements revealed RecA42 had an average translational diffusion coefficient, D20(L) = 8 +/- 2 x 10(-8) cm2/s, molecular weight, M(r) = 1.6 +/- 0.1 x 10(6), and radius of gyration, RG = 465 +/- 29 A . The smaller aggregate, RecA18, had D20(S) = 20.5 +/- 2.5 x 10(-8) cm2/s, M(r) = 7.0 +/- 0.4 x 10(5), and RG = 300 +/- 20 A . Heating RecA18 at 37 degrees C overnight resulted in conversion to a species with hydrodynamic properties indistinguishable from RecA42, called RecA18/42 . Conversion of RecA42 to RecA18 occurred almost instantaneously by 50% dilution at 38 degrees C or very slowly with incubation at 4 degrees C for at least 39 days . Self-association reactions of the three starting oligomeric states (RecA18, RecA42, and RecA18/42) induced by MgCl2 were monitored at several temperatures by dynamic light scattering . Results of these experiments provided evaluations of kinetic activation parameters of the self-association reactions . The activation parameters found for each starting oligomeric state of the protein were significantly different, revealing the variable influence of MgCl2 on the activation barriers to RecA self-association . Highly aggregated equilibrium solutions that ultimately form in solutions of each starting oligomeric species, incubated in MgCl2 at 38 degrees C for four days, were characterized by total intensity light scattering . Interpretations of these data in terms of characteristic behavior of random polymers suggests the surface morphologies of these highly associated equilibrium states formed from RecA42 and RecA18/42 are similar but contrast with that of RecA18 . Calculated values of the translational diffusion coefficient D0 were obtained for oligomeric structures modeled as helical arrays of connected monomer spheres . Best agreement with experimentally determined diffusion coefficients required that constituent monomer spheres of RecA42 have radii 33-40% larger than the monomer spheres of RecA18 . Results suggest the hydrodynamically distinct oligomeric forms of RecA may reside in conformational states with different surface exposure of hydrophobic residues, which results in substantial differences in local solvation/hydration.

Mol Microbiol, 1996 Apr, 20(1), 223 - 32
Conserved amino acids in the N- and C-terminal domains of integral membrane transporter FhuB define sites important for intra- and intermolecular interactions; Bohm B et al.; Transport of iron(III) hydroxamates across the inner membrane of Escherichia coli is mediated by a peri-plasmic binding protein-dependent transport (PBT) mechanism . FhuB, the integral membrane component of the system, is composed of covalently linked halves (FhuB{N} and FhuB{C}) which still function when present as two distinct polypeptide chains . Our analysis of two uptake-deficient FhuB derivatives provides evidence for a mechanistically novel type of functional complementation: 'domain displacement' in the cyto-plasmic membrane . Amino acid residues 60 and 426 in the FhuB polypeptide chain may define key positions that are important for FhuB{N}-FhuB{C} interaction . Furthermore, FhuB derivatives, altered in either one of their conserved regions--typical of PBT related integral membrane proteins--displayed a dominant negative effect on ferric hydroxamate transport . The experimental data suggest that the two functionally equivalent conserved regions in FhuB{N} and FhuB{C} are primarily involved in the interaction with another component of the transport system, probably FhuC.

Mol Microbiol, 1996 Apr, 20(1), 43 - 51
Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino-terminal ATP-binding domain; Rajapandi T et al.; SecA protein, the ATPase promoting translocation of proteins across the Escherichia coil inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide . In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with {alpha-(32)P}-ATP . This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein . The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral membrane forms . Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site . These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane . Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high affinity ATP-binding domain.

Antimicrob Agents Chemother, 1996 Apr, 40(4), 858 - 62
Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene; Gaurivaud P et al.; In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase . By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence) . We constructed a chimeric rpoB gene made of the E . coli rpoB gene in which the Rif region was replaced by the equivalent region from S . citri . E . coli cells harboring this chimeric gene were resistant to rifampin . Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E . coli.

Genetics, 1996 Apr, 142(4), 1379 - 82
Selection intensity for codon bias and the effective population size of Escherichia coli; Berg OG; The selection intensity for codon bias and the synonymous diversity have been used in the recent literature to estimate the effective population size of Escherichia coli . The results have varied between (10)5 and (10)8 . It is suggested here that most of this disparity can be explained by a model that accounts for the population structure of the species . Thus it is assumed that weakly selected characters, like synonymous substitutions, are selectively fixed within individual lines or colonies but spread throughout the population in an essentially neutral way when colonies replace one another . In this way, the effective population size that enters expressions for the codon bias will be that of an individual colony, which, if hitchhiking effects are considered, can be a very small number . The effective population size that appears together with the mutation rate in expressions for the synonymous diversity, on the other hand, will be related to the total number of colonies that make up the species and can be a very large number.

Protein Sci, 1996 Apr, 5(4), 719 - 28
Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: implications for domain switching; Murata LB et al.; Each catalytic (c) polypeptide chain of Escherichia coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helices . Helix 12, near the C-terminus, extends from the second domain back through the first domain, bringing the two termini close together . This helix is of critical importance for the assembly of a stable enzyme . The trimeric E . coli enzyme ornithine transcarbamoylase (OTCase) is proposed to be similar in tertiary and quaternary structure to the ATCase trimer and has a predicted alpha-helical segment near its C-terminus . In our companion paper, we have shown that this putative helix is essential for OTCase folding and assembly (Murata L, Schachman HK, 1996, Protein Sci 5:709-718) . Here, the similarity between OTCase and the ATCase trimer, which are 32% identical in sequence, was tested further by the construction of several chimeras in which various structural elements were switched between the enzymes by genetic techniques . These elements included the two globular domains and regions containing the C-terminal helices . In contrast to results reported previously (Houghton J, O'Donovan G, Wild J, 1989, Nature 338:172-174), none of the chimeric proteins exhibited in vivo activity and all were insoluble when overexpressed . Attempts to make hybrid trimers composed of c chains from ATCase and OTCase were also unsuccessful . These results underscore the complexities of specific intrachain and interchain side-chain interactions required to maintain tertiary and quaternary structures in these enzymes.

Protein Sci, 1996 Apr, 5(4), 709 - 18
Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase; Murata LB et al.; Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known . On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E . coli aspartate transcarbamoylase (ATCase) . In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers . Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains . OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions . As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix . Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase . Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions . Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces . These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.

Mol Membr Biol, 1996 Apr-Jun, 13(2), 67 - 79
Protein:protein interactions in the lipid bilayer (review); Harrison PT; At least four different types of interaction between protein transmembrane helices have been described to date . These include the use of charge-pair interactions that can play a positive or negative role in the assembly of multi-subunit complexes such as the T cell receptor, or recruit signal transducing accessory molecules in the case of some Fc receptors . Inter-helix hydrogen bonds have been shown to play an important role in the constitutive activation of certain proto-oncogenes, whereas helix:helix interfaces stabilized solely by van der Waals contacts mediated by non-polar residues also exist . The fourth type of interaction is an inter-chain disulphide linkage which is dependent on a buried charged residue . A role for glycine residues in several of these mechanisms is also suggested . In addition, the use of disulphide mapping to further explore protein:protein interactions within the lipid bilayer is discussed.

Bioseparation, 1996 Apr, 6(2), 107 - 13
Amplified expression and large-scale purification of protein L'; Murphy JP et al.; The gene fragment (PPL') encoding the functional unit of peptostreptococcus protein L was isolated using PCR and expressed in E . coli . As the gene fragment lacked its own promoter, the 5' PCR primer was designed to incorporate an Nde1 restriction site (CATATG) into the gene . This enabled the gene to be cloned in frame into an Nde1 restriction site immediately downstream of a trp promoter . To prevent read through, a stop codon was introduced into the 3' primer . Expression of PPL' was up to 27% total cell protein which compares favourably to the 0.1% total soluble cell protein obtained from the original clone of peptostreptococcus . Following a heat step homogeneous PPL' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in yields of 90%.

J Clin Microbiol, 1996 Apr, 34(4), 828 - 33
Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis; Shenai BR et al.; An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II . The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose-N-acetylgalactosamine binding lectin . This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli . Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography . The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis . Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA . The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001) . Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.

Insect Biochem Mol Biol, 1996 Apr, 26(4), 355 - 64
Presence of the Periplaneta lectin-related protein family in the American cockroach Periplaneta americana; Kawasaki K et al.; We determined the partial amino acid sequences of Periplaneta lectin, which we had purified and characterized previously from the hemolymph of the American cockroach (Periplaneta americana) {Kubo T . and Natori S . (1987) Eur . J . Biochem . 168, 75-82} . Based on these sequences, we performed PCR and found that the cDNA library of the Periplaneta fat body contained many similar, but not identical, Periplaneta lectin-related cDNAs . Analysis of the cloned cDNAs suggested that Periplaneta has a protein family, of which the periplaneta lectin and LPS binding protein purified previously are members.

Chem Biol, 1996 Apr, 3(4), 263 - 75
Two distinct binding sites for globotriaosyl ceramide on verotoxins: identification by molecular modelling and confirmation using deoxy analogues and a new glycolipid receptor for all verotoxins; Nyholm PG et al.; BACKGROUND: The Escherichia coli verotoxins (VTs) can initiate human vascular disease via the specific recognition of globotriaosyl-ceramide (Gb3) on target endothelial cells . To explore the structural basis for receptor recognition by different VTs we used molecular modelling based on the crystal structure of VT1, mutational data and binding data for deoxy galabiosyl receptors . RESULTS: We propose a model for the verotoxin 'cleft-site complex' with Gb3 . Energy minimizations of Gb3 within the 'cleft site' of verotoxins VT1, VT2, VT2c and VT2e resulted in stable complexes with hydrogen-bonding systems that were in agreement with binding data obtained for mono-deoxy analogues of Gb3 . N-deacetylated globoside (aminoGb4), which was found to be a new, efficient receptor for all verotoxins, can be favourably accommodated in the cleft site of the VTs by formation of a salt bridge between the galactosamine and a cluster of aspartates in the site . The model is further extended to explain the binding of globoside by VT2e . Docking data support the possibility of an additional binding site for Gb3 on VT1 . CONCLUSIONS: The proposed models for the complexes of verotoxins with their globoglycolipid receptors are consistent with receptor analogue binding data and explain previously published mutational studies . The results provide a first approach to the design of specific inhibitors of VT-receptor binding.

Cell Adhes Commun, 1996 Apr, 3(6), 487 - 95
The type III connecting segment of fibronectin contains an aspartic acid residue that regulates the rate of binding to integrin alpha 4 beta 1; Jongewaard IN et al.; The type III connecting segment (IIICS) within fibronectin is the major binding site for the integrin alpha 4 beta 1 . Most integrin ligands have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at position 21 . Alanine scanning mutagenesis was used to determine the amino acid residues within the intact IIICS domain required for interaction with alpha 4 beta 1 . IIICS was cloned and expressed as a fusion protein with glutathione S-transferase . This recombinant form of IIICS supports the adhesion of CHO cells that express human alpha 4 beta 1 in a cation dependent manner . Alanine scanning mutagenesis of the EILDVP sequence in recombinant IIICS demonstrated that only two of these residues are critical for adhesion of alpha 4 beta 1 expressing cells . Mutations of leucine at position 20 and aspartic acid at position 21 to alanine significantly reduced cell adhesion . Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also reduced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacement . Most importantly, we show that although the mutation of asp 21 impairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion through alpha 4 beta 1 . In comparison to wild type IIICS, the asp 21 to ala mutant supported minimal adhesion at early time points (10-30 min.), but was equivalent to wild type IIICS in supporting adhesion over one hour.

J Protein Chem, 1996 Apr, 15(3), 305 - 13
Analysis of the active center of branching enzyme II from maize endosperm; Kuriki T et al.; Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis . When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared . These putative catalytic residues are located in three different regions (regions 2-4) of the four highly conserved regions (regions 1-4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes . Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported . The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

J Protein Chem, 1996 Apr, 15(3), 291 - 304
Evidence for essential arginine residues at the active sites of maize branching enzymes; Cao H et al.; Alignment of 23 branching enzyme (BE) amino acid sequences from various species showed conservation of two arginine residues . Phenylglyoxal (PGO) was used to investigate the involvement of arginine residues of maize BEI and BEII in catalysis . BE was significantly inactivated by PGO in triethanolamine buffer at pH 8.5 . The inactivation followed a time- and concentration-dependent manner and showed pseudo first-order kinetics . Slopes of 0.73 (BEI) and 1.05 (BEII) were obtained from double log plots of the observed rates of inactivation against the concentrations of PGO, suggesting that loss of BE activity results from as few as one arginine residue modified by PGO . BE inactivation was positively correlated with {14C}PGO incorporation into BE protein and was considerably protected by amylose and/or amylopectin, suggesting that the modified arginine residue may be involved in substrate binding or located near the substrate-binding sites of maize branching enzymes I and II.

J Protein Chem, 1996 Apr, 15(3), 273 - 9
Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell; Chou WY et al.; A cDNA coding for human breast cancer cell cytosolic NADP(+)-dependent malic enzyme was obtained . This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3'-noncoding region . Comparing this nucleotide sequence with that from the normal human tissue {Loeber, G., Dworkin, M . B., Infante, A., and Ahorn, H . (1994), FEBS Lett . 344, 181-186} reveals that three nucleotides in the open reading frame and the length of 3'-noncoding region of the cDNA are different . One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure . A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally in Escherichia coli cells . Its Km value for L-malate (1.21 +/- 0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29 +/- 0.04 mM) but similar to that for the full-length recombinant enzyme (1.06 +/- 0.07 mM) . The Km values for Mn2+ and NADP+ (0.26 +/- 0.03 and 0.97 +/- 0.4 microM, respectively) are similar to those for the natural enzyme (0.12 +/- 0.02 and 1.9 +/- 0.3 microM, respectively) or the recombinant wild-type enzyme (0.56 +/- 0.04 and 0.44 +/- 0.02 microM, respectively) . A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison . The Km values for L-malate and Mn2+ of the truncated enzyme (11.2 +/- 0.9 mM and 61.2 +/- 4.6 microM, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21 +/- 0.02 mM and 1.06 +/- 0.08 microM, respectively) or the recombinant wild-type enzyme (0.25 +/- 0.01 mM and 1.48 +/- 0.05 microM, respectively) . We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.

Drug Metab Dispos, 1996 Apr, 24(4), 401 - 7
Endotoxemia in rats is associated with induction of the P4504A subfamily and suppression of several other forms of cytochrome P450; Sewer MB et al.; Bacterial lipopolysaccharide (LPS) has been previously shown to down-regulate the mRNA and protein expression of the hepatic cytochrome P450 (P450) isozymes 2C11 and 2C12 . In this study, we examined the effects of LPS on the constitutive expression of P4503A2, P4502E1, and the P4504A subfamily in the rat . Fischer 344 and Sprague-Dawley rats were each administered 1 mg/kg LPS intraperitoneally and killed for hepatic RNA and microsome isolation at different times . LPS treatment was found to suppress P4502C11, P4503A2, and P4502E1 protein and mRNA expression in both strains of rat . Total microsomal P450 levels decreased by 30%, which was smaller than the effects on the levels of individual isozymes . The magnitude of suppression exhibited in the Sprague-Dawley rats, however, seemed to be more variable than that in the F344 strain . The mRNAs of all three of the P4504A subfamily members were induced 2- to 6-fold in the F344 rat livers after LPS administration . P4504A3 protein expression increased 2-fold, whereas P4504A1/2 protein levels decreased by 30% . Lauric acid omega-hydroxylase activity increased 1.6-fold in LPS-treated Fischer 344 rats and omega-1-hydroxylase activity decreased by 38% . In the Sprague-Dawley strain, however, decreases were seen in both omega- and omega-1-hydroxylase activities after LPS treatment . Our data demonstrate that LPS administration induces P4504A subfamily mRNA and P4504A3 protein expression . Furthermore, our findings also suggest strain differences in both suppression and induction of P450s between the Sprague-Dawley and F344 rats.

J Neurovirol, 1996 Apr, 2(2), 87 - 100
Expression of multiple classes of the nuclear factor-1 family in the developing human brain: differential expression of two classes of NF-1 genes; Sumner C et al.; Nuclear factor-1 (NF-1) is a multifunctional protein that participates in both transcription and replication . NF-1 proteins exist as a family of proteins that share some common structural and functional features but also demonstrate organ and cell type specific expression . Based upon these characteristics, the family of NF-1 proteins is divided into four classes, A, B, C and D . Several NF-1 binding sites have been identified in the regulatory sequences of the human polyomavirus, JCV, which multiplies most efficiently in glial cells derived from human fetal brain . Nuclear proteins from these cultures bind specifically to these NF-1 sites . It is not known, however, which member(s) of the NF-1 family is expressed in cells susceptible to JCV infection . We have examined glial cells as well as HeLa cells, which are not permissive to JCV, for NF-1 expression . By RT-PCR analysis, all four classes of NF-1 are expressed in human fetal glial cells and HeLa cells . However, by Northern analysis the expression of class D gene is much higher in the glial cells than HeLa cells . Expression of the class C gene, first identified in HeLa cells as NF-1/CTF1, is barely detectable in glial cells but highly expressed in HeLa cells . The screening of cDNA libraries from two early human brain tissues resulted in the identification of a number of clones which appear to be related and belong to a single class of the NF-1 family, class D . Nucleotide sequence of one clone, designated NF-1/AT1, confirms this . The NF-1/AT1 protein was overexpressed in E coli and found to bind specifically to an NF-1 probe by gel shift analysis . Southern analysis of human fetal glial cells indicates that the NF-1/AT1 gene, class D, is derived from a different gene than NF-1/CTF1 . These results suggest the possibility that genes or viruses, like JCV, which use NF-1 for their expression in human brain derived cells may preferentially use the NF-1 class D protein.

Curr Opin Biotechnol, 1996 Apr, 7(2), 190 - 7
Expression of correctly folded proteins in Escherichia coli; Georgiou G et al.; Many heterologous polypeptides fail to fold into their native state when expressed in Escherichia coli; instead, they are either degraded by the cellular proteolytic machinery or accumulate in insoluble form, typically as inclusion bodies . Misfolding is a particularly vexing problem in the expression of mammalian proteins, especially those that are composed of multiple subunits, have several disulfide bonds, or contain prosthetic groups . Fortunately, bacteria exhibit a remarkable physiological plasticity that can be successfully exploited to improve protein folding . Significant yields of active heterologous proteins have been obtained through strategies that include the co-expression of homologous or heterologous folding accessory proteins, the optimization of growth conditions, and the use of fusion proteins . A flood of recent reports documenting the successful production of complex eukaryotic proteins in active form have demonstrated that bacteria can provide the proper environment for the folding of the vast majority of recombinant polypeptides.

Cell Struct Funct, 1996 Apr, 21(2), 123 - 32
Characterization of the properties of a human homologue of Escherichia coli RecQ from xeroderma pigmentosum group C and from HeLa cells; Tada S et al.; We showed that DNA-dependent ATPase Q1 (DNA helicase Q1) from xeroderma pigmentosum complementation group C (XP-C) cells elutes from FPLC Mono Q column at higher concentrations of KCl than that from other human cells (35) . We purified DNA helicase Q1 from XP-C and HeLa cells . The purified fractions of both cells contained a major polypeptide with a molecular mass of 73 kDa and had the same enzymatic properties, including salt- and temperature-sensitivity . Characterization using an anti-DNA helicase Q1 antibody indicated that this enzyme localized in the nuclei and was not modified by incorporating phosphate groups through phosphorylation and ADP-ribosylation . No interactions of DNA helicase Q1 with other proteins were indicated by immunoprecipitation of the helicase from crude extracts . No difference was observed in XP-C cells in intracellular localization of DNA helicase Q1, phosphorylation, and the interaction with other proteins as compared to HeLa cells.

Avian Dis, 1996 Apr-Jun, 40(2), 484 - 7
Surgical correction of ileus in a blue-and-gold macaw (Ara ararauna); van der Horst H et al.; A blue-and-gold macaw (Ara ararauna) was presented with a history of vomiting and production of small amounts of thin feces . A diagnosis of partial obstructive ileus was made following an oral barium-contrast study of the alimentary canal . The ileus proved to be the result of an adhesion of an intestinal loop to the abdominal wall and was successfully corrected by surgical intervention.

Avian Dis, 1996 Apr-Jun, 40(2), 417 - 24
Synovitis, osteomyelitis, and green liver in turkeys associated with Escherichia coli; Droual R et al.; Birds in seven commercial meat turkey flocks ranging in age from 8 to 11 weeks experienced lameness with swollen joints . In addition to synovitis, the most frequent lesions were swollen liver, green liver, and osteomyelitis . Different serotypes of Escherichia coli were isolated from lesions . Histopathology revealed the absence of respiratory lesions in five flocks and the presence of enteritis in at least five flocks . Hemorrhagic enteritis virus infection was implicated in six flocks by positive serology, diagnostic histopathology, and/or clinical history . Three E . coli serotypes, isolated from different types of lesions in turkeys, were inoculated intravenously into 7-wk-old poults and produced synovitis and swollen livers 3 days postinoculation . These findings suggest that the synovitis, osteomyelitis, and green liver complex is a distinct form of disease associated with E . coli, which may result from hematogenous spread of the bacteria following hemorrhagic enteritis virus infection.

Mol Biochem Parasitol, 1996 Apr, 77(1), 49 - 56
Molecular cloning, expression and characterization of a recombinant glutathione S-transferase from Echinococcus multilocularis; Liebau E et al.; We report the identification and characterization of the first cestode glutathione S-transferase (GST) cDNA sequence . A fragment of an Echinococcus multilocularis glutathione S-transferase cDNA was isolated by the polymerase chain reaction . Subsequently, a Lambda zap cDNA library prepared from mRNA from protoscolices of E . multilocularis was screened with this PCR fragment . A complete cDNA clone was isolated and the nucleotide sequence determined . Analysis of the E . multilocularis GST-deduced amino acid sequence indicates that it is clearly related to the mammalian mu-class GSTs . The E . multilocularis GST cDNA was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein . The 25.5-kDa enzyme subunit was purified to homogeneity using glutathione-sepharose chromatography . Gel filtration demonstrated that this GST is enzymatically active as a homodimer . The recombinant enzyme had conjugating activity with organic hydroperoxides and with members of the trans,trans-2,4 alkadienal and trans-2-alkenal series, which are secondary products of lipid peroxidation.

Trop Gastroenterol, 1996 Apr-Jun, 17(2), 70 - 6
Determinants of symptomatic giardiasis in childhood; Rajeshwari K et al.; Giardia has been frequently implicated as a causative agent for acute as well as chronic diarrheal diseases in children . The present study was aimed at exploring the determinants of manifestations of Giardiasis in childhood, in relation to various host and parasite related factors . A total of 200 children with acute (100), chronic (50) or without (50) diarrhea in last 15 days were recruited for the study and evaluated with regards to nutritional status, serum immunoglobulins, secretory IgA levels, presence of Giardia in stool/duodenal aspirate/duodenal biopsy specimen and for associated infections . Lysates from acute giardiasis cases were further studied for zymodeme (banding) pattern . After correlation of all investigations, humoral immune defect in the host was found to be the major determinant of whether the Giardial infestation would be symptomatic or not, while associated bacterial infections and zymodeme patterns were not found to be important in determining the pathogenicity or presentation of giardiasis.

Vaccine, 1996 Apr, 14(6), 511 - 20
Combination of human cytomegalovirus recombinant immediate-early protein (IE1) with 80 nm cationic biovectors: protection from proteolysis and potentiation of presentation to CD4+ T-cell clones in vitro; Prieur E et al.; We have shown in a previous study that the proliferative CD4+ T-cell response to the regulatory immediate-early protein IE1 was a major component of the overall anti viral response in human cytomegalovirus (HCMV) seropositive blood donors . This viral antigen may be valuable in subunit vaccine design, since anti IE1 CD4+ T cells might provide help for production of antibodies and cytotoxic T lymphocytes (CTL) responses, and could take part in the control of viral infection . Preliminary to the elaboration of future vaccine formulations, we developed immunogenic complexes resulting from the combination of a purified recombinant protein derived from the fusion of Escherichia coli glutathione-S-transferase (GST) and a large C-terminal fragment (e4) of IE1, with new 80 nm cationic synthetic particles called Biovectors . We have shown that the antigen GST-e4 was stably complexed to vectors and that, contrary to the soluble form, it was protected from proteolysis in cell culture medium . By confocal microscopy we observed that the synthetic vectors were internalized by lymphoblastoid B cells, providing a significant enhancement of antigen delivery in antigen presenting cells (APC) . Indeed, we demonstrated that the previous combination of antigen with particles, significantly enhanced the proliferation of specific CD4+ T-cell clones directed against IE1 in vitro, when either HLA-matched isolated peripheral blood mononuclear cells or EBV transformed B cell lines were used as APC . The relevance of these observations to the use of these new vectors for vaccine design against HCMV is discussed.

J Am Coll Nutr, 1996 Apr, 15(2), 180 - 5
Nutritional impact and ultrastructural intestinal alterations in severe infections due to enteropathogenic Escherichia coli strains in infants; Fagundes-Neto U et al.; OBJECTIVE: Enteropathogenic Escherichia coli (EPEC) strains are able to adhere to human intestinal tissue inducing a typical lesion causing dissolution of the brush border membrane and loss of microvillus structure at sites of bacterial attachment . The presence of these lesions can provoke perpetuation of diarrhea associated with malabsorption of the nutrients and nutritional aggravation . In this paper we report the nutritional impact of severe EPEC gastroenteritis in infants in a small bowel ultrastructural study . METHODS AND RESULTS: Two infants aged 3 months and one 4 month old infant with profuse watery diarrhea lasting less than 6 days were studied . After rehydration therapy, the patients were fed a cow's milk formula and since food intolerance was diagnosed, a protein-hydrolisate, lactose-free formula was introduced . This dietary modification was successful, diarrhea ceased, the patients were discharged and followed up for 30 days . The following EPEC strains were identified in the stools and in the jejunal secretion: O111ab:H2, O119:H6 and O18ab:H14 . A small bowel biopsy was performed and the electron microscopic study revealed bacteria tightly adhered to the apical portion of the enterocyte and effacement of the microvilli . These lesions were more prominent in the areas where bacteria were present . CONCLUSION: The patients underwent an acute nutritional aggravation due to food intolerance, but the introduction of a protein-hydrolisate, lactose-free formula, allowed prompt cessation of diarrhea and nutritional recovery.

Jpn J Pharmacol, 1996 Apr, 70(4), 285 - 90
The mediators involved in endotoxin-induced vascular permeability increase in the rat skin and their interactions; Ueno A et al.; The injection of lipopolysaccharide (LPS) from E . Coli into the dorsal skin of rats caused a dose-dependent increase in vascular permeability as measured by the extravasation over a 40-min period of intravenously injected dye . This increase caused by LPS was attenuated by pretreatment with the bradykinin (BK) receptor antagonist HOE140, the selective platelet-activating factor (PAF) antagonist TCV309, and by combined treatment with mepyramine and methysergide . Combined treatment with HOE140 and TCV309 resulted in further suppression than that achieved with a single treatment alone . By the simultaneous pretreatment with all antagonists, the response was almost totally abolished . On the other hand, indomethacin also inhibited the response induced by LPS, but not those induced by BK and PAF itself . A small dose of BK or histamine synergistically potentiated the effect of PAF when simultaneously injected . These results suggest that BK, PAF, histamine/serotonin and prostaglandins are involved in the LPS-induced increase in vascular permeability, where PAF, in addition to its direct action, potentiates the response to BK and histamine, and prostaglandins potentiate the actions of other mediators without its direct action.

Bioorg Khim, 1996 Apr, 22(4), 243 - 51
{Human tumor necrosis factor mutants: preparation and some properties}; Shingarova LN et al.; Using polymerase chain reaction, a number of mutant genes encoding human tumor necrosis factor (TNF-alpha) with amino acid substitutions and a deletion were obtained . The mutant proteins (muteins) contained point mutations R32H, A33S, F144L, I118M, and I118A; double mutation R32H-F144L; and deletion of four amino acid residues 67-70 . The mutant genes were expressed in E . coli under the control of constitutive promoters . A simple purification method for the muteins was developed and their physicochemical properties were studied . All the muteins obtained, except F144L and I118A, were shown by CD and cross-linking to from a spatial structure similar to that of the native TNF-alpha . The collection of muteins was characterized by their biological activity . Mutants R32H and A33S exerted a decreased cytotoxicity against murine fibroblast cell line L929, whereas point mutant F144L and double mutant R32H-F144L were essentially inactive.

Zhonghua Yi Xue Za Zhi, 1996 Apr, 76(4), 254 - 7
{The role of TNF alpha, IL-1 beta and MIP-1 alpha in LPS-induced organ injury}; Qiu H et al.; OBJECTIVE: To investigate the role of inflammatory cytokines in endotoxemia models and to explore the possible anti-cytokine therapy of endotoxemia . METHODS: TNF alpha in plasma was measured by ELISA, and the mRNA of cytokines was assessed by slot blot analysis . RESULTS: LPS-induced TNF alpha release was in a dose:dependent manner in human whole blood . Dexamethasone (> or = 10(-8)mol/L) exhibited inhibitory effect on TNFa release . Ibuprofen 10(-7)-10(-9) mol/L had inhibitory effect on TNFa production, whereas 10(-3)-10(-4)mol/L stimulated TNFa release . The rat model of acute lung injury was made by LPS intraperitoneal injection . Both dexamethasone and ibuprofen that injected at 1 hour before LPS injection could decrease the contents of Evans blue in the lung (t 2.80 and 7.31 respectively, P < 0.05 vs LPS control) . After LPS administered, there was a progressive up regulation in transcripts of TNF alpha, IL-1 beta and MIP-1 alpha in whole lung homogenates of rats . The mRNA peaked at 2, 6, 12 hours respectively . The TNF alpha, IL-1 beta and MIP-1 alpha mRNA levels decreased markedly when dexamethasone or ibuprofen given at 1 hour before LPS injection . CONCLUSION: TNF alpha, IL-1 beta and MIP-1 alpha play an essential role in the inflammatory response . LPS-induced acute lung injury may be prevented by dexamethasone and ibuprofen which have inhibitory effect on the gene expression of cytokines in the lung.

Genetika, 1996 Apr, 32(4), 523 - 31
{Localization of RecA-like proteins in preparations of spread nuclei of mouse spermatocytes at meiotic prophase I}; Loseva EF et al.; Earlier, polyclonal antibodies to Escherichia coli RecA protein were used to identify immunologically related proteins in meiotic spermatocytes of different eukaryotes . At least one such protein proved to be a component of the synaptonemal complex (SC) {1} . Subsequent experiments on localization of RecA-like antigens in SCs of spermatocytes were performed by indirect immunocytochemical methods and electron microscopy, which showed that RecA-like protein(s) at early leptotene are largely associated with chromatin . During SC formation (at leptotene and zygotene), they are found in both lateral elements and the central space of SC . In some cases, RecA-like proteins are associated with SC substructures that resemble recombination nodules . When spermatocytes enter late diplotene, Rec-A-like proteins cease to be detected in SC structures.

J Biochem (Tokyo), 1996 Apr, 119(4), 703 - 10
Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function; Ohmae E et al.; To elucidate the role of a flexible loop (residues 64-72) in the stability and function of Escherichia coli dihydrofolate reductase, glycine-67 in this loop was substituted by site-directed mutagenesis with seven amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val) . The circular dichroism spectra suggested that the confirmation of the native structure was affected by the mutations in both the presence and absence of NADPH . The free energy change of unfolding by urea decreased in the order of G67A > G67S > or = wild-type > or = G67D > G67T > G67C > or = G67L > G67V . The steady-state kinetic parameters for the enzyme reaction, Km and kcat, were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by the deuterium isotope effect on the enzyme activity . These results suggest that site 67 in the flexible loop, being very far from the active site, plays an important role in the stability and function of this enzyme . The characteristics of the mutations were discussed in terms of the modified flexibility of the native structure, compared with the results of mutations at site 121 in another flexible loop.

J Biochem (Tokyo), 1996 Apr, 119(4), 667 - 73
Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization; Itoh M et al.; Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines . Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution . The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner . The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7 . Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7 . The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions . The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors . The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state . Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.

J Biochem (Tokyo), 1996 Apr, 119(4), 653 - 8
Characterization and comparison of synthetic immobile and mobile Holliday junctions; Shida T et al.; Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresis, ultraviolet absorption and circular dichroism (CD) spectroscopy . Four 24-mer deoxyribonucleotides formed stable immobile and mobile HJs in 0.1 M NaCl at 5 muM strand concentration at room temperature . However, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used . A comparison of the melting profiles of the HJs with those of the duplexes corresponding to the arms of four-way junctions indicated that the thermal stability of the HJ was similar to that of the individual arm and the cooperativity of the melting behavior of the HJ was relatively higher than that of the individual arm duplex . The Tms of the mobile HJs containing 4, 6, 8, and 10 base-pair homologous cores at junctions were essentially identical with that of the immobile HJ of the same size . There is a tendency that the HJ containing a larger homologous core region becomes more resistant to thermal denaturation . The addition of divalent metal cations, Mg2+ and Ca2+, to the solutions of the HJs raised their melting temperatures . The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ depended primarily upon the DNA sequence flanking the junction . The RuvC protein binds to the immobile and mobile HJs, regardless of the presence and the size of the homologous core at the junction.

Hybridoma, 1996 Apr, 15(2), 109 - 16
Epitope mapping of new monoclonal antibodies recognizing distinct human FcRII (CD32) isoforms; Weinrich V et al.; The class II Fc gamma receptors are widely distributed on cells of the immune system . Nevertheless, the exact cell type distribution of the FcRII isoforms is still unclear because of the lack of appropriate antibodies that discriminate between the various isoforms . In this study we describe the generation and characterization of three monoclonal antibodies (MAbs) raised against recombinant human FcRIIb2 as well as a synthetic peptide (amino acids 30-39) of this receptor . Analyses of the isoform specificity of these antibodies using ELISA and Western blots revealed that the MAbs II1A5 (mIgG1) and ID2.7 (mIgM) are pan FcRII antibodies recognizing all known FcRII isoforms . In contrast, the MAb II8D2 (mIgG1) specifically reacts with FcRIIb but not with FcRIIa . The observed antibody reactivities could be confirmed by examination of the exact epitopes using overlapping 15-mer peptides spanning the entire FcRIIb2 . So far these antibodies are the only ones described that detect FcRII in Western blots . Moreover, they can be used to analyze the cellular FcRII isoform distribution at the protein level, which was otherwise not possible.

J Vet Med Sci, 1996 Apr, 58(4), 365 - 7
Passage of chicken egg yolk antibody treated with hydroxypropyl methylcellulose phthalate in the gastrointestinal tract of calves; Ikemori Y et al.; Two types of chicken egg yolk antibody samples for oral passage trials in calves were prepared: (1) hydroxypropyl methylcellulose phthalate (HPMCP) antibody powder (HAP)--a powder produced by spray-drying a supernatant obtained after precipitation of lipids from egg yolk with HPMCP and (2) control antibody power (CAP)--a powder produced from an antibody solution with HPMCP . Antibody activity and pattern of distribution of both antibody preparations in the gastrointestinal tract of calves were compared by enzyme-linked immunosorbent assay . At 2 hr post administration, anti-K99 fimbrial antibodies from both the CAP and the HAP were detected in the abomasum of calves with titers of 1:128 and 1:256, respectively . However, at 4 hr, anti-K99 fimbrial titers of the CAP and the HAP were reduced to 1:2 and 1:64, respectively, due to digestion in the abomasum . These results indicated that the egg yolk antibody powder with HPMCP was more resistant against gastric juice in the stomach, thereby, ensuring a transfer of functional antibodies to the small intestine of calves after oral administration.

FEMS Immunol Med Microbiol, 1996 Apr, 13(4), 317 - 23
A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies; Dubreuil JD et al.; STb is a heat-stable enterotoxin elaborated by enterotoxigenic Escherichia coli strains associated with weaning piglets and is responsible for diarrhoea in those animals . The maltose binding protein (MBP) of E . coli was used as a carrier for STb, a poorly immunogenic molecule . Constructions were produced where the gene coding for mature STb toxin (MBP-STb) and a fragment of the gene spanning the major epitopic region of STb (AA8-AA30) (MBP-STb2) were fused to malE gene coding for MBP . The fusion proteins accumulated in the periplasm and were detected with a polyclonal antibody raised against the purified toxin . MBP-STb induced secretion in the biological model whereas MBP-STb2 was non-toxic . Immunization of rabbits evoked an antibody response to STb for these two fusion proteins . However, only MBP-STb elicited antibodies that effectively neutralized the toxicity of pure STb toxin as determined in the rat loop assay.

Int J Pept Protein Res, 1996 Apr, 47(4), 311 - 21
Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli; Andersson C et al.; A new variant of human growth hormone was recently found {Pavlu, B . & Gellerfors, P . (1993) Bioseparation 3, 257-265} . We report here the identification and the structural determination of this variant . The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography . The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH . This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz . Cys182-Cys189 . Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical . Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH . A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e . m/z 617.7 and 682.9 . These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide . Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV . Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed . Using these data all protons were assigned . The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19 . CD spectroscopy data were also in agreement with the above results . Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH . The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore . The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.

Electrophoresis, 1996 Apr, 17(4), 781 - 3
Isolation of proteins and nucleic acids by electrophoresis on disposable gel columns; Costa MA et al.; A simple and cheap one-step method to isolate proteins or nucleic acids by electrophoresis in disposable gel columns is reported . A disposable syringe was modified to host a gel column and an elution chamber . Starting from a crude extract of E . coli, the laboratory-made devise allowed the isolation of the maltose binding protein (MBP) fused to a recombinant allergenic molecule with a molecular mass of 58 kDa, from a mixture of several proteins . Also, plasmid DNA could be isolated from a mixture containing chromosomal DNA and RNA, avoiding the use of organic solvents . Electrophoresis was performed at 150 V, 35 degrees C, pH 8.0 and 8.3 for protein and DNA, respectively . The protein or the DNA obtained showed a yield of 80% and a purity grade of 90%, as estimated by densitometry.

Protein Eng, 1996 Apr, 9(4), 365 - 70
Production and characterization of anti-human interferon gamma receptor antibody fragments that inhibit cytokine binding to the receptor; Bridges A et al.; Three single-chain antibody fragments that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R), and inhibit the binding of human IFN gamma, have been produced in Escherichia coli . These fragments are derived from murine anti-receptor monoclonal antibodies, and comprise the variable heavy (VH) domain linked to the variable light (VL) chain through a 15 amino acid linker {(GGGGS)3} . Using surface plasmon resonance technology (BIAcore), the soluble proteins were shown to retain a high affinity for recombinant IFN gamma R, and by radioimmunoassay to possess a high inhibitory activity towards IFN gamma-binding to human Raji cells . The antibody fragments most likely recognize epitopes that overlap the cytokine binding site on the receptor surface . Attempts to dissect further the antibodies to isolated VH- and VL-chains and to synthetic linear and cyclic peptides derived from the individual complementarity determining regions failed to afford fragments with significant IFN gamma R binding affinity . Nevertheless, these native-like variable region fragments and petidomimetics derived from them are of interest in the design of novel IFN gamma R antagonists.

Protein Eng, 1996 Apr, 9(4), 345 - 52
Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2 . Substitution of histidine by 1,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH; Beiboer SH et al.; The effect of the substitution of the active site histidine 48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acid analogue in porcine pancreas phospholipase A2 (PLA2) was studied . TAA was introduced biosynthetically using a his-auxotrophic Escherichia coli strain . To study solely the effect of the substitution of the active site histidine, two nonessential histidines (i.e . His17 and His115) were replaced by asparagines, resulting in a fully active mutant enzyme (His-PLA2) . In this His-PLA2 the single histidine as position 48 was substituted by TAA with an incorporation efficiency of about 90%, giving a mixture of His-PLA2 and TAA-PLA2 . Based on the charge difference at acidic pH, both forms could be separated by FPLC, allowing for the purification of TAA-PLA2 free from His-PLA2 . At pH 6, TAA-PLA2 has a fivefold reduced activity compared with His-Pla2 . This reduced activity paralells a reduced rate of covalent modification with p-nitrophenacyl bromide of TAA-PLA2 compared with His-PLA2 . Competitive inhibition gave comparable IC50 values for WT-PLA2, His-PLA2 and TAA-PLA2 . These results indicate that the reduction in activity is not caused by a different affinity for the substrate, but more likely results from a reduced kcat value in TAA-PLA2 . The enzymatic activities for native and mutant PLA2s were measured at different pH values . For WT-PLA2 and His-PLA2 the activity is optimal at pH 6 and is strongly deminished at acidic pH, with no observable activity at pH 3 . In contrast, TAA-PLA2 is as active at pH 3 as at pH 6 . Most likely, the decrease in activity observed for WT-PLA2 and His-PLA2 is caused by the protonation of the active site His48, which is the general base involved in the activation of the nucleophilic water molecule . In TAA-PLA2, however, the active site residue TAA48 is unprotonated at both pH 3 and 6 as a result of the low pKa of TAA compared with histidine.

Virus Res, 1996 Apr, 41(2), 141 - 51
Site-directed mutagenesis of the double-stranded RNA binding domain of bacterially-expressed sigma 3 reovirus protein; Wang Q et al.; The affinity of the reovirus sigma 3 protein for double-stranded RNA (dsRNA) is well established, and efforts have been made to identify the amino acids involved in this property . In the present study, we further examined the importance of two basic amino acids motifs, located in the carboxy-terminal third of the protein . Mutants, previously characterized in COS cells, were expressed in bacterial cells using the pET expression system . The capability of the different mutants to interact with dsRNA was then determined by the binding of radiolabeled dsRNA to proteins resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters . It appears that the most carboxy-terminal motif is absolutely required for the binding but the second motif also contributes to this property . However, only the carboxy-terminal motif is required for normal binding upon removal of the amino-terminal domain of the protein by proteolytic cleavage, a procedure previously shown to increase dsRNA-binding . The basic charges in both motifs are important, while breaking of their potential to adopt an alpha helical configuration does not affect binding efficiency . Furthermore, alanine substitution of a single basic amino acid in the carboxy-terminal motif can be sufficient to strongly reduce the binding of dsRNA to the protein . Altogether, these data suggest that basic amino acids of the sigma 3 carboxy-terminal motif are directly involved in dsRNA binding, while the other basic motif may contribute by preventing an inhibitory effect of the amino-terminal portion of the protein.

Glycoconj J, 1996 Apr, 13(2), 159 - 66
Influence of phospholipid chain length on verotoxin/globotriaosyl ceramide binding in model membranes: comparison of a supported bilayer film and liposomes; Arab S et al.; The importance of the surrounding lipid environment on the availability of glycolipid carbohydrate for ligand binding was demonstrated by studying the influence of phosphatidylcholine fatty acid chain length on binding of verotoxins (VT1 and VT2c) to their specific cell surface receptor, globotriaosylceramide (Gb3) in the presence of auxiliary lipids both in a microtitre plate surface bilayer film and in a liposome membrane model system . In the microtitre assay, both VT1 and VT2c binding to Gb3 was increased as a function of decreasing PC acyl chain length likely resulting in increased Gb3 exposure . In the liposome assay VT1 binding was similarly modulated, however the effect of VT2c binding was more complex and did not follow a simple function of increased carbohydrate exposure . Earlier work established that C22:1 and C18:1Gb3 fatty acid homologues were the preferred Gb3 receptor containing liposomes, but in C14PC liposomes, binding to C22:1Gb3 (but not C18:1Gb3) was elevated such that this Gb3 species now became the preferred receptor for both toxins . This change in verotoxin/Gb3 homologue binding selectivity in the presence of C14PC did not occur in the microtitre bilayer format . These results are consistent with our proposal that these toxins recognize different epitopes on the Gb3 oligosaccharide . We infer that relative availability of these epitopes for toxin binding in an artificial bilayer is influenced not only by the exposure due to the discrepancy between the fatty acyl chain lengths of Gb3 and PC, but by the physical mode of presentation of the bilayer structure . Such acyl chain length differences have a more marked effect in a supported bilayer film whereas only the largest discrepancies affect Gb3 receptor function in liposomes . The basis of phospholipid modulation of glycolipid carbohydrate accessibility for receptor function is likely complex and will involve phase separation, gel/liquid crystalline transition, packing and lateral mobility within the bilayer, suggesting that such parameters should be considered in the assessment of glycolipid receptor function in cells.

Bioorg Med Chem, 1996 Apr, 4(4), 553 - 6
Synthesis of 2'-deoxyuridine 5'-(alpha,beta-imido) triphosphate: a substrate analogue and potent inhibitor of dUTPase; Persson T et al.; The dUDP analogue, 2'-deoxyuridine 5'-(alpha,beta-imido)diphosphate (dUPNP) was synthesized . The corresponding triphosphate analogue (dUPNPP) was prepared by enzymic phosphorylation of dUPNP using the enzyme pyruvate kinase and phosphoenolpyruvate as the phosphate donor . This method was successful in phosphorylating the imidodiphosphate analogue of 2'-deoxythymidine (dTPNP) to 2'-deoxythymidine 5'-(alpha, beta-imido)triphosphate (dTPNPP), in contradiction to a previous report . The properties of dUPNPP have been tested using the enzyme dUTPase from Escherichia coli . This enzyme, having a crucial role in nucleotide metabolism, is strictly specific for its substrate (dUTP) and catalyzes the hydrolysis of the alpha, beta-bridge, resulting in dUMP and pyrophosphate . Replacement of the alpha, beta-bridging oxygen in dUTP with an imido group resulted in a nonhydrolyzable substrate analogue and a potent competitive inhibitor of dUTPase (Ki = 5 microM) . The analogue prepared (dUPNPP) may be utilized in crystallographic studies of the active site of dUTPase to provide knowledge about specific interactions involved in substrate binding and as a parental compound in design of dUTPase inhibition for medical purposes.

Toxicon, 1996 Apr, 34(4), 490 - 5
A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culture bioassay in combination with a water-soluble tetrazolium salt; Hamasaki K et al.; A tissue culture bioassay, using the mouse neuroblastoma cell line (Neuro2A), was improved to provide a simple and sensitive bioassay for TTX or sodium channel-blocking toxins (SCB) . The water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetraz olium, monosodium salt (WST-1), was applied to replace the time-consuming and subjective cell-counting procedure of the cells with automatic measurement, using a microplate reader . It was also confirmed that this method is directly applicable to bacterial culture supernatants, with the precaution of possible interference.

Vet Microbiol, 1996 Apr, 49(3-4), 235 - 41
CNF producing Escherichia coli isolated from cattle in Northern Ireland; Burns AL et al.; Tissue culture assays were used to investigate the incidence of cytotoxic necrotising factors (CNFs) 1 and 2 in Escherichia coli strains from cattle . E . coli cultures were obtained from faeces collected from 223 cases of diarrhoea and from 113 healthy animals . In addition, strains cultured from 62 cases of mastitis, 66 cases of septicaemia and 68 cases of abortion were also investigated . E . coli producing CNF 1 or 2 were identified in all sample groups except for the abortion cases . Comparable levels of CNF1 strains were present in E . coli from the faces of diarrhoeic (4%) and healthy faeces (4.4%) whereas lower levels of CNF2 were identified in the faeces from diarrhoeic animals (19.3%) in comparison with healthy animals (30.9%) . One CNF1 producing strain was identified among the E . coli isolated from mastitis samples, while 3% and 10.6% of septicaemic strains were positive for CNF1 and 2, respectively . Serogrouping of CNF isolates did not reveal the association of any particular serogroups with the different conditions.

J Radiol, 1996 Apr, 77(4), 271 - 4
{Pseudotumor form of focal acute pyelonephritis}; Pelage JP et al.; Two cases of focal acute pyelonephritis presenting with an unusual pseudo-tumoral appearance on CT are reported . The lesions exhibited a focal intra-parenchymal homogeneous round-shape mass without any other parenchymal or perirenal fat CT abnormalities . The final diagnosis of focal acute pyelonephritis was confirmed on following CT scans which demonstrated the total disappearance of the lesions in both cases . This particular appearance of acute focal pyelonephritis which mimicked a renal tumor should be clearly individualized in the terminology based on CT findings.

Mol Microbiol, 1996 Apr, 20(2), 435 - 47
Reciprocal regulation of the differentiation of Myxococcus xanthus by Pkn5 and Pkn6, eukaryotic-like Ser/Thr protein kinases; Zhang W et al.; Myxococcus xanthus contains a large family of genes encoding eukaryotic-like serine/threonine kinases . Among them, two genes, pkn5 and pkn6, are divergently located on the chromosome and share a 46 bp promoter region between their transcription initiation sites, as determined by RNA protection . Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein . Its membrane topology was determined using the Pkn6-PhoA fusion protein in Escherichia coli, which has a single transmembrane domain with the N-terminal domain in the cytoplasm and the C-terminal domain outside the cytoplasmic membrane . Both proteins, when expressed in E . coli, were autophosphorylated: Pkn5 only at Ser, and Pkn6 at both Ser and Thr . In M . xanthus, both genes are expressed constitutively throughout the life cycle, with slight increases at an early stage of development . Most strikingly, a pkn5-deletion strain forms fruiting bodies much faster than the wild-type strain, while a pkn6-deletion strain develops slower than the wild-type strain . These results, together with the fact that the pkn5-deletion strain is able to form fruiting bodies on semi-rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M . xanthus growth and development . Furthermore, Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase that negatively regulates M . xanthus development.

Mol Microbiol, 1996 Apr, 20(2), 375 - 84
Co-operative binding of two Trp repressor dimers to alpha- or beta-centred trp operators; Gunes C et al.; The alpha-centred trp operator binds one dimer of the Trp repressor, whereas the beta-centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992) . The Trp repressor with a Tyr-Gly-7 substitution binds almost as well as the wild-type Trp repressor to the alpha-centred trp operator, but it does not bind to the beta-centred trp operator . This confirms that Tyr-7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993) . Further experiments with alpha-centred trp operator variants showed that positions +/-1 of the alpha-centred trp operators play a crucial role in tetramerisation . The two innermost base pairs of the alpha-centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it . However, substitutions in these positions (T-A to G-T) effectively transform the alpha-centred trp operator into a beta-centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator . Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a beta-centred trp operator.

Mol Microbiol, 1996 Apr, 20(2), 351 - 60
DNA supercoiling depends on the phosphorylation potential in Escherichia coli; van Workum M et al.; ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli . Independent of whether the ATP/ ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio . The experiments were performed under well-defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e . using a minimal salts medium with succinate as the sole free-energy and carbon source, and in the presence or absence of ammonia as the nitrogen source . The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number . The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation . Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling . This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.

Mol Microbiol, 1996 Apr, 20(2), 325 - 37
A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus; Stone KD et al.; Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA) . LA is dependent upon the presence of 50-70 MDa plasmids, termed EPEC adherence factor (EAF) plasmids . Expression of an EAF plasmid-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype . TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA . While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10 kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes . The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described . The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis . However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred . This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein . Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E . coli strain, but is insufficient for LA . This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E . coli strain with a defined set of genes . The BFP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.

Mol Microbiol, 1996 Apr, 20(2), 313 - 23
EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells; Kenny B et al.; Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhoea . EPEC mediates several effects on host epithelial cells, including activation of signal-transduction pathways, cytoskeletal rearrangement along with pedestal and attaching/effacing lesion formation . It has been previously shown that the EPEC eaeB (espB) gene encodes a secreted protein required for signal transduction and adherence, while eaeA encodes intimin, an EPEC membrane protein that mediates intimate adherence and contributes to focusing of cytoskeletal proteins beneath bacteria . DNA-sequence analysis of a region between eaeA and eaeB identified a predicted open reading frame (espA) that matched the amino-terminal sequence of a 25 kDa EPEC secreted protein . A mutant with a non-polar insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement . These phenotypes were complemented by a cloned espA gene . The espA mutant is also defective for invasion . It is concluded that espA encodes an EPEC secreted protein that is necessary for activating epithelial signal transduction, intimate contact, and formation of attaching and effacing lesions, processes which are central to pathogenesis.

Mol Microbiol, 1996 Apr, 20(2), 295 - 311
Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1; Pedersen LB et al.; The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1 . One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2, is functionally virtually uncharacterized . In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented . By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed . Furthermore, Hc2 was found to strongly inhibit translation and transcription in vitro . Our results imply that DNA condensation is not the only function of Hc2.

Br J Pharmacol, 1996 Apr, 117(8), 1792 - 6
Effect of cyclo-oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide-induced neutrophil infiltration in mouse lung; Goncalves de Moraes VL et al.; 1 . The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents . In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response . 2 . Inhalation of LPS led to a time and dose-dependent increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice . Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS-sensitive mouse strain C3H/HeN, but was absent in the LPS-resistant strain C3H/HeJ . Intranasal administration of murine recombinant TNF-alpha also triggered neutrophil recruitment . 3 . One hour after inhalation of LPS, half of the maximal level of TNF-alpha was measured in the BALF, but only a few neutrophils were detected at this time . The peak TNF-alpha concentration was reached at 3 h, when the neutrophil amount started to increase . At 24 h, maximal neutrophil number was found in the BALF and TNF-alpha was no longer present . 4 . Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF-alpha production; (b) anti TNF-alpha antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF-alpha production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF-alpha production . 5 . It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS-induced TNF-alpha synthesis and neutrophil recruitment.

Br J Pharmacol, 1996 Apr, 117(7), 1530 - 4
Regulation of tumour necrosis factor production by adrenal hormones in vivo: insights into the antiinflammatory activity of rolipram; Pettipher ER et al.; 1 . The role of adrenal hormones in the regulation of the systemic and local production of tumour necrosis factor (TNF alpha) was examined in male Balb/c mice . 2 . Intraperitoneal injection of 0.3 mg E . coli lipopolysaccharide (LPS, 0111:B4) led to high levels of circulating TNF alpha without stimulating TNF alpha production in the peritoneal cavity . Systemic production of TNF alpha in response to LPS was increased in adrenalectomized animals and in normal animals treated with the beta-adrenoceptor antagonist, propranolol . The glucocorticoid antagonist, RU 486, did not modify systemic TNF alpha production . These results indicate that systemic TNF alpha production is regulated by adrenaline but not by corticosterone . 3 . When mice were primed with thioglycollate, TNF alpha was produced in the peritoneal cavity in response to low dose LPS (1 micrograms) . The levels of TNF alpha in the peritoneal cavity were not enhanced by adrenalectomy or by treatment with either propranolol or RU 486, indicating local production of TNF alpha in the peritoneal cavity is not regulated by adrenaline or corticosterone . 4 . The phosphodiesterase type IV (PDE-IV) inhibitor, rolipram, inhibited both the systemic production of TNF alpha in response to high dose endotoxin (ED50 = 1.3 mg kg-1) and the local production of TNF alpha in the peritoneal cavity in response to low dose endotoxin (ED50 = 9.1 mg kg-1) . In adrenalectomized mice there was a slight reduction in the ability of rolipram to inhibit the systemic production of TNF alpha (ED50 = 3.3 mg kg-1) while the ability of rolipram to inhibit the local production of TNF alpha in the peritoneal cavity was virtually abolished (24% inhibition at 30 mg kg-1) . The glucocorticoid antagonist, RU 486, also reduced the ability of rolipram to inhibit local TNF alpha production while propranolol was without effect . 5 . Systemic treatment with rolipram increased the plasma concentrations of corticosterone in normal mice but not in adrenalectomized mice indicating that rolipram can cause adrenal stimulation in vivo . 6 . In summary, these data indicate that systemic production of TNF alpha in response to high dose endotoxin is controlled differently from the local production of TNF alpha in response to low dose endotoxin . The systemic production of TNF alpha is regulated by catecholamines, but not by corticosterone, while the local production of TNF alpha in the peritoneal cavity is not regulated by basal levels of either catecholamines or corticosterone . 7 . These data also show that the ability of rolipram to inhibit the local production of TNF alpha is dependent on the release of corticosterone from the adrenal glands.

Br J Pharmacol, 1996 Apr, 117(7), 1449 - 56
The role of lipocortin-1 in dexamethasone-induced suppression of PGE2 and TNF alpha release from human peripheral blood mononuclear cells; Sudlow AW et al.; 1.Lipocortin-1 and its N-terminal derivatives exert potent inhibitory actions in various models of acute inflammation . The present study examined the ability of lipocortin (LC)-1 to suppress the release of the acute pro-inflammatory mediators, tumour necrosis factor (TNF alpha) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin-1 beta (rhIL-1 beta) . 2 . LPS (10 micrograms ml-1) stimulated release of TNF alpha and PGE2 from PBMC was significantly inhibited by (4 h) co-incubation of the cells with 10(-6) M dexamethasone (Dex), but not with 10(-9) M to 10(-7) M of a N-terminal fragment (amino acids 1-188) of recombinant human LC-1 (LC-1 fragment) . However, Dex suppression of LPS-stimulated TNF alpha and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC-1 fragment (1:10,000 dilution) was included in the medium . rhIL-1 beta (5 x 10(-8) M)-stimulated release of TNF alpha and PGE2 from PBMC (after 18 h) was abolished by co-incubation of the cells with 10(-7) M LC-1 fragment . 3 . After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti-LC-1 fragment antibody (which showed to cross-reactivity with human annexins 2 to 6) . Dex caused no increase in immunoreactive (ir)LC-1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC-1-like immunoreactivity . This was accompanied by the appearance of irLC-1 in the extracellular medium . 4 . The results of the present study implicate endogenous LC-1 in glucocorticoid suppression of TNF alpha and PGE2 release from human PBMC and suggest an extracellular site of action for LC-1 . LC-1 may also inhibit rhIL-1 beta-stimulated TNF alpha and PGE2 secretion from PBMC.

Br J Pharmacol, 1996 Apr, 117(7), 1421 - 6
Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory derivatives with gastrointestinal-sparing properties; Cirino G et al.; 1 . The effects of novel nitric oxide-releasing nonsteroidal anti-inflammatory compounds (NO-NSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line, J774 . 2 . LPS-induced nitrite production was markedly attenuated by the nitroxybutylester derivatives of flurbiprofen (FNBE), aspirin, ketoprofen, naproxen, diclofenac and ketorolac, with each compound reducing accumulated nitrite levels by > 40% at the maximum concentrations (100 micrograms ml-1) used . 3 . Further examination revealed that nitrite production was inhibited in a concentration-dependent (1-100 micrograms ml-1) manner by FNBE which at 100 micrograms ml-1 decreased LPS-stimulated levels by 63.3 +/- 8.6% (n = 7) . The parent compound flurbiprofen was relatively ineffective over the same concentration-range, inhibiting nitrite accumulation by 24 +/- 0.9% (n = 3) at the maximum concentration used (100 micrograms ml-1) . 4 . FNBE reduced LPS-induced nitrite production when added to cells up to 4 h after LPS . Thereafter, FNBE caused very little or no reduction in nitrite levels . Furthermore NO-NSAIDs (100 micrograms ml-1) did not inhibit the metabolism of L-{3H}-arginine to citrulline by NO synthase isolated from LPS-activated macrophages . 5 . Western blot analysis demonstrated that NO synthase expression was markedly attenuated following co-incubation of J774 cell with LPS (1 microgram ml-1; 24 h) and FNBE (100 micrograms ml-1; 24 h) . Thus taken together, these findings indicate that NO-NSAIDs inhibit induction of NO synthase without directly affecting enzyme activity . 6 . In conclusion our results indicate that NO-NSAIDs can inhibit the inducible L-arginine-NO pathway, and are capable of suppressing NO synthesis by inhibiting expression of NO synthase . The clinical implications of these findings remain to be established.

Trends Microbiol, 1996 Apr, 4(4), 147 - 53
Role of verotoxin receptors in pathogenesis; Lingwood CA; Verotoxin-globotriaosyl ceramide (Gb3) binding is the linchpin in disease induced by verotoxin-producing Escherichia coli (VTEC), and defines cell sensitivity, tissue tropism, mode of systemic transport, specific cytotoxic activity and internal routing within sensitive cells . Binding explains the epidemiology of renal pathology, which may follow VTEC infection . Lipid heterogeneity of Gb3 is important in binding, and may define a growth-related signal transduction pathway used by verotoxin.

Biokhimiia, 1996 Apr, 61(4), 745 - 54
{Analysis of the effect of replacing Lys(-20) in the alkaline phosphatase signal peptide on secretion of this enzyme}; Karamysheva ZN et al.; The effect of substitutions for the positively charged Lys(-20) in the N-terminal domain of the E . coli alkaline phosphatase signal peptide on enzyme secretion has been studied . Mutant alkaline phosphatases were obtained by the amber-suppressor method . An amber mutation was introduced in the appropriate position of the alkaline phosphatase gene using oligonucleotide-directed mutagenesis . This was followed by mutant protein synthesis in E . coli strains producing amber-suppressor tRNAs specific for Tyr, Gly, Ala, Glu, Phe, His, Cys, and Pro . All the mutant proteins can by translocated through the cytoplasmic membrane and form in the periplasm a molecule possessing an enzymatic activity . However, some amino acid substitutions decrease the rate of protein maturation their effect depends not only on the charge of the amino acid residue but also on its nature . Thus, introduction of positively charged . His and the polar uncharged Tyr is without effect, while negatively charged Glu and hydrophobic Ala, Phe and Pro residues as well as Gly and Cys have an inhibiting action . The results obtained testify to the importance of the signal peptide terminal domain primary structure in secretion.

Immunol Cell Biol, 1996 Apr, 74(2), 151 - 8
Molecular cloning and characterization of tumor necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula; Wedlock DN et al.; Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood . We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha . Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF-alpha and the possum cDNA, derived from spleen RNA, identified by RT-PCR . The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE) . The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF-alpha genes . The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively . Cell extracts prepared from transformants and the purified GST/TNF-alpha fusion protein exhibited cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells . The induction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers . Macrophages cultured in the presence of LPS showed enhanced transcription of TNF-alpha mRNA . This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.

J Biomol Struct Dyn, 1996 Apr, 13(5), 727 - 39
Flexible loop in the structure of S-adenosylmethionine synthetase crystallized in the tetragonal modification; Fu Z et al.; S-Adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, E.C.2.5.1.6.) plays a central metabolic role in all organisms . MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi) from ATP and L-methionine . AdoMet is the primary methyl group donor in biological systems . MAT from Escherichia coli was crystallized in the tetragonal modification with space group P4(3)2(1)2 using the same conditions as previously yielded crystals of the hexagonal system {Takusagawa, et al., (1996), J . Biol . Chem . 171, 136-147}, except for the crystallization temperature . The structure has been determined by molecular replacement at 3.2 A resolution . The overall structure of the tetrameric MAT in the tetragonal modification is essentially the same as the structure found in the hexagonal modification . However there are two remarkable differences between the structures of two modifications . One is the contents in the active sites (holoform vs . apo-form), and the other is the conformation of the flexible loop over the active site (open vs . closed) . These differences in the crystal structures are caused solely by the difference in crystallization temperatures (26 degrees C vs . 4 degrees C) . We have interpreted the structural data obtained from the X-ray analyses in conjunction with the results of the mechanistic and sequencing studies in terms of possible dynamic motion of the flexible loop . When a substrate/product binds in the active site (hexagonal modification), the loop becomes disordered, apparently due to flexibility at the entrance of the active site as if it acts as a "mobile loop" during the catalytic reaction . On the other hand, when the temperature is decreased, the dynamic motion of the flexible loop may be reduced, and the loop residues enter the active site and close its entrance (tetragonal modification) . Thus, the active site of the tetragonal modification is empty despite the crystals being grown in mother liquor containing a large concentration of phosphate (100 mM) . There is no significant displacement of amino acid residues in the active site between the holo and apo forms, suggesting that the flexible loop plays an important role in determination of the contents in the active site . Since the functionally important amino acid residues in the active site are all conserved throughout various species, the structures of the active sites and the mechanism of the catalysis are probably essentially identical in the enzymes from a wide range of organisms . However, the substrate KM and Vmax values of MATs from various species are distributed over a wide range . The amino acid residues in the flexible loop regions are poorly conserved throughout various species . Therefore, the wide differences in catalysis rates of MATs from various speeches may be due to the differences in the composition of the flexible loop.

J Wildl Dis, 1996 Apr, 32(2), 199 - 208
Immunotoxicity studies in mink (Mustela vison) chronically exposed to dietary bleached kraft pulp mill effluent; Smits JE et al.; The immunotoxic potential of bleached kraft pulp mill effluent (BKME) to cell-mediated immunity in mink (Mustela vison) was investigated October 1993 through May 1994 . For 26 weeks, 20 mink were fed a diet based upon fish caught within 6 km downstream of a bleached kraft mill in Saskatchewan, Canada . Water for this group contained 25% softwood-run BKME . Twenty control mink were fed nutritionally matched diets based upon fish from lakes receiving no municipal or industrial effluent and tap water . Using in vitro and in vivo immunotoxicity assays, the proliferative response of mink peripheral blood mononuclear cells (PBMC) to mitogens was optimal, at 72 hr with 10 micrograms/ml Concanavalin A, 1/80 dilution pokeweed mitogen, and 1/80 dilution phytohemagglutinin . Bacterial cell wall Escherichia coli lipopolysaccharide did not stimulate mitosis of the mink PBMC . No difference (P < 0.05) in PBMC proliferation was seen between the control and BKME-exposed mink with any of the mitogens used . Delayed type hypersensitivity (DTH), a cell mediated response, was assessed in mink vaccinated with live bacille Calmette-Guerin (BCG) and then challenged by intradermal toe web injection with 200 micrograms of sonicated BCG approximately 6 weeks later . The DTH response in the BKME-exposed mink was impaired based upon assessment using skin thickness measurements, histopathological assessment and image analyzer technology . This decreased response is evidence for suboptimal immune function associated with BKME exposure, which could affect the competitive fitness of piscivorous mammals naturally exposed to BKME.

Invest Radiol, 1996 Apr, 31(4), 194 - 203
Computed tomography of experimental liver abscesses using a new liposomal contrast agent; Dick A et al.; RATIONALE AND OBJECTIVES . Evaluation of computed tomographic enhancement characteristics of a new liposomal contrast agent (liposomal iodixanol {LI}) in a pyogenic liver abscess model in 17 rabbits . METHODS . Eight to 14 days after abscess induction (Escherichia coli), density-time curves were calculated for regions of interest in liver, abscess wall and center, spleen, portal vein, abdominal aorta, inferior vena cava, and kidney . Images were obtained every minute between 1 and 10 minutes, every 5 minutes between 15 and 60 minutes, and 75 minutes after 200 mg/kg LI application (group A: 7 rabbits) and after 600 mg/kg iopentol application (group B: 10 rabbits), and 90, 105, and 120 minutes after LI . RESULTS . The abscess wall-liver contrast after LI lasted from 10 to more than 120 minutes with a maximum of 30 delta Hounsfield Units (HU) at 45 minutes . For iopentol, the abscess wall-liver contrast lasted from 2 to 7 minutes with a maximum of 8 delta HU at 5 minutes . The abscess wall-center contrast after LI lasted from 1 to more than 120 minutes with a maximum of 112 delta HU at 40 minutes . For iopentol, the abscess wall-center contrast lasted from 1 to 75 minutes with a maximum of 79 delta HU at 1 minute . The liver-portal vein contrast after LI lasted from 1 to more than 120 minutes with a maximum of 100 delta HU at 20 minutes . For iopentol, the liver-portal vein contrast lasted from 1 to 8 minutes with a maximum of 38 delta HU at 2 minutes . An abscess wall was detected in a higher percentage of the LI images (86% LI, 56% iopentol), and images in the LI group correlated better with histopathology . CONCLUSIONS . The diagnostic value of LI exceeds that of iopentol in terms of overall abscess contrast and duration of the diagnostic interval . The higher hepatic vessel contrast allows better abscess localization.

Shock, 1996 Apr, 5(4), 304 - 10
Pulmonary surfactant function following endotoxin: effects of exogenous surfactant treatment; Picone A et al.; In a porcine model of endotoxin-induced adult respiratory distress syndrome (ARDS) we tested the hypothesis that the severity of lung injury would vary with the concentration of endotoxin and that reestablishment of normal surfactant function with exogenous surfactant would vary with the severity of lung injury . The therapeutic effects of exogenous surfactant treatment on pulmonary surfactant function have varied greatly in animal models of ARDS . This has created discrepancies in the literature that may be due in part to a difference in the severity of the pulmonary lesion . Yorkshire pigs were anesthetized, placed on a ventilator, and surgically prepared for hemodynamic and lung function measurements . Pigs received either 25 (25LPS) or 50 (50LPS) micrograms/kg of Escherichia coli lipopolysaccharide (LPS) followed by exogenous surfactant (SURF, 100 mg/kg) instillation, and were randomized into five groups: Control = sham LPS (n = 4); 25LPS (n = 6); 50LPS (n = 6); 25LPS + SURF (n = 5); and 50LPS + SURF (n = 6) . Treatments were followed by histological and surfactant function evaluation . Histological evaluation showed the hallmarks of ARDS . Pulmonary surfactant function assessed by surfaced tension minimum (Ymin) was significantly (P < .05) elevated in both the 25LPS (20.2 +/- 2, dyne/cm) and 50LPS (19 +/- 3, dyne/cm) groups as compared with the Control group (10 +/- 1, dyne/cm) . Exogenous surfactant reduced Ymin in the 25LPS + SURF group (9 +/- 2 dyne/cm, p < .05 vs . 25LPS) but not in the 50LPS + SURF group (20 +/- 1 dyne/cm, p < .05 vs . Control and 25LPS + SURF) . Surfactant treatment was more effective in reestablishing normal surfactant function in animals subjected to a low dose of endotoxin, compared with animals receiving a higher dose.

Shock, 1996 Apr, 5(4), 284 - 8
Dibutyryl cAMP improves systemic vasoconstriction caused by endotoxin in dogs; Yanase T et al.; We studied whether dibutyryl cyclic adenosine monophosphate (DbcAMP), which freely penetrates into the cells, improves systemic vasoconstriction caused by endotoxin in dogs . Thirteen anesthetized dogs were randomized into three groups . The endotoxin (ETX) group (n = 5) received only Escherichia coli endotoxin (3 mg.kg-1, intravenously) . The ETX + DbcAMP group (n =5) received DbcAMP (6 mg.kg-1, intravenously) 30 min before the administration of endotoxin . The DbcAMP group received the same dose of DbcAMP 30 min after administration of saline . In the ETX group, systemic blood pressure and cardiac index significantly decreased, and systemic vascular resistance significantly increased, while in the ETX + DbcAMP group, increases in systemic and pulmonary vascular resistances after the administration of endotoxin were attenuated . DbcAMP did not cause hemodynamic changes in normal dogs . Plasma concentrations in thromboxane B2 in the ETX group were higher than in the ETX + DbcAMP group . Also, the change in plasma cyclic AMP concentrations showed a good logarithmic correlation with the change in plasma thromboxane B2 concentrations after the administration of endotoxin (r = .908, log (delta T x B2) = -.002* (delta cAMP) + 3.786) . We conclude that DbcAMP improves systemic vasoconstriction caused by endotoxin in dogs . The beneficial mechanism of DbcAMP on systemic vasoconstriction after the administration of endotoxin may be partially due to inhibition of thromboxane B2.

Shock, 1996 Apr, 5(4), 274 - 9
Comparison of the capacity of rhTNF-alpha and Escherichia coli to induce procoagulant activity by baboon mononuclear cells in vivo and in vitro; Li A et al.; The procoagulant activity of mononuclear cells (MNCs) may play an important role in the disseminated intravascular coagulation seen in septic shock . This study compares the capacity of Escherichia coli (E . coli) and recombinant human TNF-alpha (rhTNF-alpha) to induce procoagulant activity by baboon MNCs . In vivo studies showed that MNC procoagulant activity was significantly increased at T + 120 min after LD100 E . coli infusion into baboons . Most of this procoagulant activity was attributable to tissue factor . In contrast, a bolus infusion of rhTNF-alpha (150 micrograms/kg) and a monoclonal antibody to activated protein C (2 mg/kg) did not induce any increase of MNC procoagulant activity at T + 120 min even though the plasma TNF-alpha level was 10 times higher than that seen following infusion of E . coli . In vitro studies showed that E . coli at concentrations comparable to that observed in the vivo study and LPS at a concentration of 2.5 ng/mL induced more intense tissue factor expression by both human and baboon monocytes than rhTNF-alpha in the concentrations ranging from 10 to 1,000 ng/mL . These results suggest that TNF-alpha alone is not sufficient to induced noticeable MNC procoagulant activity, at least, in the early stage of this septic shock model.

Am J Vet Res, 1996 Apr, 57(4), 477 - 82
Intramammary defense against infections induced by Escherichia coli in cows; Paape MJ et al.; OBJECTIVE--To examine Escherichia coli lipopolysaccharide (LPS) effects on expression of CD14 and CD18 cell surface receptors and lectin/carbohydrate-mediated nonopsonic phagocytosis of E coli . DESIGN--Cell isolation, monoclonal antibody, phagocytosis, and flow cytometric studies . ANIMALS--4 clinically normal lactating Holstein cows for studies on CD14 and CD18, and 2 for phagocytosis studies . PROCEDURE--Binding of CD14 and CD18 monoclonal antibodies to blood and milk neutrophils and mononuclear leukocytes was studied by flow cytometry before and after intramammary injection of LPS, and nonopsonic phagocytosis of E coli by blood neutrophils was determined . Presence of intracellular CD14 was determined after in vitro incubation of neutrophils in skimmed milk and after fixation and permeabilization of freshly isolated neutrophils . RESULTS--Before LPS injection, percentages of blood neutrophils and large mononuclear (LMO) cells expressing CD14 averaged 3 and 63% and 68 and 35% for mammary neutrophils and LMO cells, respectively . After LPS injection, CD14 was only detected on blood and mammary LMO cells (61 and 25%); receptor expression increased by 1.8- and threefold, respectively . In vitro incubation of neutrophils in skimmed milk increased the percentage of neutrophils expressing CD14 . The number of blood neutrophils staining positive for CD14 increased after permeabilization of the plasma membrane, which was blocked by unlabeled anti-CD14 monoclonal antibodies . Before LPS, percentages of blood neutrophils and LMO cells expressing CD18 averaged 93 and 95% and was 88 and 55% for mammary neutrophils and LMO cells, respectively . After LPS, percentages of mammary neutrophils and LMO cells expressing CD18 increased to 100 and 95%, respectively . Expression of CD18 was 2.6-fold higher for mammary neutrophils before injection of LPS, compared with blood neutrophils, either before or after LPS . In absence of opsonins, neutrophils with adherent and phagocytosed E coli averaged 83 and 14% . CONCLUSIONS--LPS modulated expression of CD14 and CD18 and lectin-carbohydrate interactions mediated nonopsonic phagocytosis of E coli . An intracellular pool of CD14 exists in bovine neutrophils and is capable of translocating to the cell surface . CLINICAL RELEVANCE--Development of methods to maximize expression of CD14 receptors on mammary neutrophils involved in production of tumor necrosis factor-alpha, and nonopsonic phagocytosis could result in reducing prevalence of mastitis in dairy cows.

Plant Mol Biol, 1996 Apr, 31(1), 87 - 100
A novel kinesin-like protein with a calmodulin-binding domain; Wang W et al.; Calcium regulates diverse developmental processes in plants through the action of calmodulin . A cDNA expression library from developing anthers of tobacco was screened with 35S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins . Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained . The TCK1 cDNA encodes a protein with 1265 amino acid residues . Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception . Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes . Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner . In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region . The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins . The motor domain has ATPase activity that is stimulated by microtubules . Southern blot analysis revealed that TCK1 is coded by a single gene . Expression studies indicated that TCK1 is expressed in all of the tissues tested . Its expression is highest in the stigma and anther, especially during the early stages of anther development . Our results suggest that Ca2+/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

Plant Mol Biol, 1996 Apr, 31(1), 69 - 76
Cloning of a cDNA encoding a 3-dehydroquinate synthase from a higher plant, and analysis of the organ-specific and elicitor-induced expression of the corresponding gene; Bischoff M et al.; cDNA clones for all enzymes of the prechorismate pathway of higher plants have previously been cloned, with the exception of the second enzyme of the pathway, 3-dehydroquinate synthase . Here we describe the isolation of a cDNA encoding a 3-dehydroquinate synthase from tomato which was identified by complementing a 3-dehydroquinate synthase-deficient Escherichia coli strain with a tomato cDNA library . The deduced amino acid sequence contains a putative N-terminal plastid-specific transit peptide, and the sequence of the mature enzyme resembles those of the corresponding bacterial enzymes more than of the fungal enzymes . Sequence identity was even higher between the tomato and E . coli sequences than between the E . coli and other known bacterial sequences . The abundance of 3-dehydroquinate synthase transcripts differ in the organs of tomato plants analyzed . In cultured tomato cells, the abundance of 3-dehydroquinate synthase transcripts increased 9-fold within 4 to 5 h of elicitor treatment.

Pediatr Nephrol, 1996 Apr, 10(2), 203 - 5
Cluster of cases of haemolytic uraemic syndrome due to unpasteurised cheese; Deschenes G et al.; A cluster of four patients (1 girl, 3 boys) from a French village (2,000 inhabitants) had acute haemolytic uraemic syndrome (HUS) between March 1992 and May 1993 . All had prodromes with fever and diarrhoea, then acute renal failure, anaemia, schistocytosis and thrombocytopenia . Peritoneal dialysis was carried out in three children (duration 3-12 days) . The verotoxin VT2 gene was identified by polymerase chain reaction in the stools of two children . Some days prior to the diarrhoea, all children had eaten a cheese made with unpasteurised mixed cows' and goats' milk from the same farm . A case control study showed that the occurrence of HUS was linked to the consumption of this milk product (P = 0.006) . The VT 2 gene was isolated from the cheese and from the stools of goats and cows from the farm, but not from the stools of farm employees.

Zentralbl Veterinarmed A, 1996 Apr, 43(2), 93 - 101
Studies on in vivo endotoxin plasma disappearance times in cattle; Andersen PH et al.; Endotoxin plasma disappearance (EPDT) times were determined by a modified Limulus amoebocyte lysate (LAL) assay technique after the intravenous administration of 25 micrograms E . coli 055:B5 endotoxin per kg b.w . to 22 Jersey cows . Clinically healthy cows (n = 6) cleared endotoxin from the plasma within 30 min . Cows pretreated with flunixin meglumine (n = 6) had 2-3 times longer plasma disappearance times, while cows pretreated with phenylbutazone (n = 6) had plasma disappearance times which were 6-12 times longer than the healthy control group . A fourth group comprised clinical cases of spontaneously developed hepatic lipidosis (n = 4) . None of these cows were able to clear the injected endotoxin dose and one died before the end of the experiment . The acute phase response, described by leukocyte and thrombocyte counts and plasma glucose and zinc concentrations, was not statistically different between the four groups.

J Med Virol, 1996 Apr, 48(4), 329 - 38
Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C; Pilot-Matias TJ et al.; The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently . The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized . Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E . coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins . Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis . Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses . An antigenic region was also identified in the putative core protein of GB virus B . Many of the clones identified originally as encoding antigenic proteins were quite large . To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot . Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened . These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.

J Nucl Med, 1996 Apr, 37(4), 673 - 9
Technetium-99m-white blood cell-specific imaging agent developed from platelet factor 4 to detect infection; Moyer BR et al.; We have developed a leukocyte-avid, 99mTc-labeled peptide (P483H) as a potential imaging agent for infection . P483H contains the heparin-binding region of platelet factor-4 (PF-4) and a lysine-rich sequence for rapid renal clearance . Technetium-99m-P483H was evaluated for its ability to selectively label white blood cells (WBCs) in vitro and to detect focal E . coli infections in rabbits . METHODS: Technetium-99m-P483H was incubated with citrated whole human blood, layered onto WBC isolation media and subjected to density gradient centrifugation to measure WBC-associated radioactivity . Indium-111-WBCs and 99mTc-gluceptate were used as controls . In the in vivo model, E . coli infected rabbits were imaged and necropsied 4 hr after administration of 99mTc-P483H . Infected and contralateral control muscles were evaluated for %ID, %ID/g, Imax (muscle sample showing the highest uptake, i.e., %ID/g) and Imax-to-blood and Imax-to-control muscle ratios . Indium-111-WBCs, 111In-DTPA, 131I-albumin (HSA), 99mTc-nanocolloid, 67Ga and 99mTc-gluceptate were evaluated as in vivo controls . RESULTS: Technetium-99m-P483H associated predominantly with WBCs in vitro, and 99m-Tc-P483H provided high contrast images of infection in vivo . In vitro, 73% of 99mTc-P483H radioactivity was associated with WBCs . Technetium-99m-P483H outperformed 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate with an infection Imax average of 0.062 %ID/g (+/- 0.029; n = 48) . Technetium-99m-P483H also outperformed all controls, including 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate . The Imax-to-blood and Imax-to-control muscle ratios for 99mTc-P483H averaged 3.1 (+/- 2.4) and 26.8 (+/- 16.8), respectively, and again outperformed all controls . CONCLUSION: Technetium-99m-P483H associates predominantly with WBCs in vitro and identified focal infections in vivo within 4 hr versus conventional imaging agents . Additionally, the agent showed rapid blood clearance and exclusive renal excretion, which provides a clear abdominal field for imaging abdominal infections.

Immunology, 1996 Apr, 87(4), 633 - 41
In vivo treatment with anti-interleukin-13 antibodies significantly reduces the humoral immune response against an oral immunogen in mice; Bost KL et al.; Interleukin-13 (IL-13) is a cytokine which significantly enhances the proliferation and differentiation of B lymphocytes . We therefore evaluated its role in the formation of a humoral immune response in vivo . Upon oral immunization with the B subunit of Escherichia coli heat-labile enterotoxin (LT-B), rapid up-regulation of IL-13 mRNA expression in the mesenteric lymph nodes of LT-B intubated mice occurred . This result suggested that IL-13 might be involved in the formation of a mucosal antibody response against LT-B if this cytokine was in fact secreted . To test this possibility, the coding region for murine IL-13 was cloned into the pFLAG-1 expression vector . Recombinant murine IL-13 was purified from bacterial lysates and used as an immunogen to produce polyclonal anti-IL-13 antibodies . Groups of BALB/c mice treated in vivo with anti-IL-13 antibody 2 days before and on the day of oral immunization with LT-B had significantly reduced intestinal IgA and serum IgG and IgA anti-LT-B antibody responses when compared to mice treated with control antibody . Furthermore, groups of mice primed with LT-B and then treated with anti-IL-13 antibody prior to oral immunization with a second dose of LT-B also had significantly reduced intestinal IgA and serum IgG and IgA anti-LT-B antibody titres compared to controls . In vitro LT-B restimulation experiments using splenic mononuclear leucocytes isolated from LT-B primed mice treated with anti-IL-13 antibody demonstrated decreased expression of IL-4 and IL-13 mRNA and decreased IL-4 secretion when compared to controls . Together these results demonstrate an important role for IL-13 in the formation of a humoral immune response at mucosal surfaces.

Plant Cell Physiol, 1996 Apr, 37(3), 355 - 62
The seed-specific transcription factor VP1 (OSVP1) is expressed in rice suspension-cultured cells; Nakagawa H et al.; A seed-specific transcriptional regulator, VP1, is required for the induction of ABA-regulated genes that include Lea (late embryogenesis abundant protein) genes . Although the induction of one rice Lea gene, Osem, by ABA is normally restricted to seed tissues, we found that the expression was strongly induced by ABA in the Oc line of rice suspension-cultured cells . Since this observation suggested that rice VP1 (OSVP1) protein or a functionally similar protein might be expressed in the cultured cells, we analyzed the expression of Osvp1 in these cells at both the mRNA and the protein level, we detected Osvp1 mRNA and OSVP1 protein in the cultured cells at levels similar to or higher than those in developing embryos . In the cultured cells, neither the level of total cellular OSVP1 nor that of nuclear OSVP1 protein was affected by ABA . Based on the results, the mechanism for the transcriptional regulation of VP1-dependent ABA-inducible genes is discussed.

Plant Cell Physiol, 1996 Apr, 37(3), 313 - 23
Identification of the chlB gene and the gene product essential for the light-independent chlorophyll biosynthesis in the cyanobacterium Plectonema boryanum; Fujita Y et al.; We cloned a 6.0-kb HindIII fragment from the cyanobacterium Plectonema boryanum using the chloroplast chlB (ORF513) gene of the liverwort (Marchantia polymorpha) as a probe . An open reading frame (ORF508) encoding a polypeptide of 508 amino acid residues was found within the nucleotide sequence of the 4,437-bp HindIII-EcoRV subfragment . The deduced amino acid sequence of ORF508 shows very high similarity to that encoded by the liverwort chlB gene (72.7%) . A mutant, YFB14, in which ORF508 was inactivated by the insertion of a kanamycin-resistance cartridge, was unable to synthesize chlorophyll, accumulating protochlorophyllide in darkness while synthesizing chlorophyll normally in the light . Thus, the chlB gene is the third gene that is essential for the light-independent reduction of protochlorophyllide . The other two genes are chlL and chlN, and the results suggest that the light-independent protochlorophyllide reductase consists of at least three subunits, which are encoded by chlL, chlN and chlB . Using an antiserum prepared against a ChlB-6xHis fusion protein expressed in Escherichia coli, we detected a protein with an apparent molecular weight of 58,000 in the membrane fraction of the cyanobacterium . These results indicate that either the cytoplasmic or thylakoid membranes could be the site of the light-independent reduction of protochlorophyllide.

Plant Cell Physiol, 1996 Apr, 37(3), 293 - 8
Identification of the product of ndhA gene as a thylakoid protein synthesized in response to photooxidative treatment; Martin M et al.; A 76 amino acid sequence of NDH-A (the protein encoded by plastid ndhA gene) from barley (Hordeum vulgare L.) was expressed as a fusion protein with beta-galactosidase in E . coli . The corresponding antibody generated in rabbits was used to investigate localization, expression and synthesis in vitro of NDH-A . NDH-A was identified as a 35 kDa polypeptide localized in thylakoid membrane . Western blots shows a large increase in NDH-A levels when barley leaves were incubated under photooxidative conditions, which was more pronounced in mature-senescent leaves than in young leaves . Immunoprecipitation of the {35S}methionine labelled proteins, synthesized in vitro by isolated chloroplasts, demonstrated the synthesis in chloroplasts of the NDH-A 35 kDa polypeptide when barley leaves had been incubated under photooxidative conditions . The results indicate that ndh genes may be involved in the protection of chloroplasts against photooxidative stress, particularly in mature-senescent leaves.

Biochem J, 1996 Apr 1, 315 ( Pt 1), 71 - 5
Characterization of the hydroxymethylglutaryl-CoA lyase precursor, a protein targeted to peroxisomes and mitochondria; Ashmarina LI et al.; We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes . HL contains a 27-residue N-terminal mitochondrial targeting sequence which in cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif . Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader . To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties . pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL . For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL . Purified pHL is similar to mHL in K(m) for HMG-CoA (44.8 microM), k(cat) (6.3 min(-1)) and pH optimum (9.0-9.5) . However, the quaternary structures of pHL and mHL differ . On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA), pHL behaves as a monomer whereas mHL migrates as a dimer . We conclude that the HL percursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization on pHL.

Biochem J, 1996 Apr 1, 315 ( Pt 1), 249 - 56
Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes; Moore PS et al.; L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids . To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides . The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein . During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney . Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer . Moreover, the Vmax was twice that of the protein purified from tissue . On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme . Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences . The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal . When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm . However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm . Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes . The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin . The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm . A putative working model is presented to explain this behaviour.

Biochem J, 1996 Apr 1, 315 ( Pt 1), 15 - 9
Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem; Doyle WA et al.; An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and pro-sequence regions of Lip isoenzyme H8 (Lip) has been successfully overexpressed in Escherichia coli . The recombinant protein (LipP*) was sequestered in inclusion bodies . The reduced-denatured polypeptide has been purified by differential solubilization, and the active enzyme recovered after controlled in vitro refolding (albeit in low yield), by glutathione-mediated oxidation of disulphides, in a folding medium containing an intermediate concentration of urea, Ca2+, and haem . The procedure is analogous to that previously described for the production of active recombinant horseradish peroxidase (HRP-C*) from inclusion-body material . It is quite possible, therefore, that this type of procedure may be suitable for the recovery of most, if not all, active recombinant peroxidases . The resultant LipP* has spectral characteristics identical with that of the native enzyme as isolated from Phanerochaete chrysosporium . Its specific activity measured in the standard veratryl alcohol (VA) assay was 39 micromol of VA oxidized/min per mg of protein, a value which compares extremely favourably with that of the native enzyme (36 micromol of VA/min per mg) . Although levels of active enzyme obtained are not yet as high as in the case of HRP-C* (1% conversion of crude inactive LipP* polypeptide into pure fully active Lip), it is envisaged that further refinement of the expression/folding/activation procedures will provide sufficient protein for biophysical characterization of both the wild-type and site-directed mutants.

Biochem J, 1996 Apr 1, 315 ( Pt 1), 103 - 12
Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis; Elborough KM et al.; In the oilseed rape Brassica napus there are two forms of acetyl-CoA carboxylase (ACCase) . As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena . This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level . Six classes of nuclear encoded oilseed rape BCCP cDNA were clones, two of which contained the entire coding region . The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences . We also report the cloning of a second type II ACCase component from oilseed rape, the beta-carboxyltransferase subunit (betaCT), which is chloroplast-encoded . Northern analysis showed that although the relative levels of BCCP and betaCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development . At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera . With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast . This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

Biol Bull, 1996 Apr, 190(2), 173 - 87
Endogenous beta-galactosidase activity in the larval, pupal, and adult stages of the fruit fly, Drosophila melanogaster, indicates need for caution in lacZ fusion-gene studies; Schnetzer JW et al.; Beta-galactosidase activity is known to exist in Drosophila melanogaster, but a detailed analysis of the tissue-specific patterns of activity has not previously been reported . Such an analysis is of particular interest because Drosophila is commonly used for making transformants that carry fusion genes in which the E . coli beta-galactosidase gene, lacZ, is used as a reporter gene . When these transformants are analyzed for beta-galactosidase activity by using chromogen X-gal staining, the method does not distinguish true fusion-gene activity from endogenous beta-galactosidase activity or from the beta-galactosidase activity of bacterial contaminants . Therefore, detailed maps of endogenous beta-galactosidase activity in this organism would help to prevent errors in data interpretation and would indicate which stages were most appropriate for experiments with the lacZ transformants . We have constructed such maps by applying X-gal staining methods to serial frozen sections and whole mounts of larval, prepupal, pupal, and adult stages of D . melanogaster reared under axenic conditions . Results showed endogenous beta-galactosidase activity in a variety of organs including the larval intestine, spiracles, lymph glands, cellular epidermis, and eye-antenna imaginal discs; the pupal cellular epidermis, lymph glands, imaginal tissues, fat body, and spiracle; and the adult pericardial cells, thoracic nephrocytes, ventriculus, and reproductive system . The good correlation between staining and metamorphic remodeling and phagocytic activity indicates that endogenous beta-galactosidase is physiologically interesting.

Am J Hum Genet, 1996 Apr, 58(4), 712 - 21
Uroporphyrinogen decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria; Moran-Jimenez MJ et al.; A deficiency in uroporphyrinogen decarboxylase (UROD) enzyme activity, the fifth enzyme of the heme biosynthetic pathway, is found in patients with sporadic porphyria cutanea tarda (s-PCT), familial porphyria cutanea tarda (f-PCT), and hepatoerythropoietic porphyria (HEP) . Subnormal UROD activity is due to mutations of the UROD gene in both f-PCT and HEP, but no mutations have been found in s-PCT . Genetic analysis has determined that f-PCT is transmitted as an autosomal dominant trait . In contrast, HEP, a severe form of cutaneous porphyria, is transmitted as an autosomal recessive trait . HEP is characterized by a profound deficiency of UROD activity, and the disease is usually manifest in childhood . In this study, a strategy was designed to identify alleles responsible for the HEP phenotype in three unrelated families . Mutations of UROD were identified by direct sequencing of four amplified fragments that contained the entire coding sequence of the UROD gene . Two new missense mutations were observed at the homoallelic state: P62L (proline-to-leucine substitution at codon 62) in a Portuguese family and Y311C (tyrosine-to-cysteine substitution at codon 311) in an Italian family . A third mutation, G281E, was observed in a Spanish family . This mutation has been previously described in three families from Spain and one from Tunisia . In the Spanish family described in this report, a paternal uncle of the proband developed clinically overt PCT as an adult and proved to be heterozygous for the G281E mutation . Mutant cDNAs corresponding to the P62L and Y311C changes detected in these families were created by site-directed mutagenesis . Recombinant proteins proved to have subnormal enzyme activity, and the Y311C mutant was thermolabile.

Leuk Res, 1996 Apr, 20(4), 333 - 41
Detection of in vivo differentiation of murine WEHI-3B D+ leukemia cells transfected with the lac-Z marker gene using two-color flow cytometry; Hayashi M et al.; The in vivo induction of the differentiation of murine WEHI-3B D+ myelomonocytic leukemia cells was measured by flow cytometry, simultaneously staining leukemia cells for the marker exogenous beta-galactosidase and for differentiation by the antigen Mac-1 (CD11b/CD18) . The WEHI-3B D+ leukemia cells were transfected with the E . coli lac-Z gene by electroporation and subclones that constitutively expressed high levels of the lac-Z gene product beta-galactosidase were established . Flow cytometric analyses of cells in the peritoneal cavities of mice bearing leukemia cells showed that cells continued to express beta-galactosidase for at least 14 days, and they were distinguishable from host-derived cells in vivo by their expression of the transfected gene . Simultaneous determination of the beta-galactosidase activity and Mac-1 content of cells in the peritoneal cavities of mice revealed that administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the mice enhanced the expression of Mac-1 antigen by beta-galactosidase-positive cells . The results demonstrate that G-CSF may have clinical potential as a therapeutic differentiating agent, and that flow cytometric analysis provides a useful in vivo system to evaluate the therapeutic potential of agents capable of inducing terminal differentiation.

J Virol, 1996 Apr, 70(4), 2611 - 4
Mutational analysis of the vaccinia virus E3 protein defines amino acid residues involved in E3 binding to double-stranded RNA; Ho CK et al.; Alanine-substitution mutations were targeted to 14 amino acid residues within the double-stranded (ds) RNA binding motif (dsRBM) of the vaccinia virus E3 protein . Substitutions at six positions--Glu-124, Phe-135, Phe-148, Lys-167, Arg-168, and Lys-171--caused significant reductions in dsRNA binding . These six residues are conserved in the two dsRBMs for which structural information is available (Escherichia coli RNase III and Drosophila melanogaster staufen) and in many other members of the dsRBM protein family . Residues we show to be important for dsRNA binding by vaccinia virus E3 map to the same face of the dsRBM structure and are thus likely to compose part of the RNA binding site.

J Virol, 1996 Apr, 70(4), 2605 - 10
Polyprotein processing in Southampton virus: identification of 3C-like protease cleavage sites by in vitro mutagenesis; Liu B et al.; A genomic clone of the small, round-structured virus Southampton virus (SV) was constructed from a set of overlapping PCR amplicons . Sequence analysis confirmed the absence of mutations and accurate ligation of the PCR products . The SV cDNA was cloned into a vector for in vitro production of RNA and subsequent translation by rabbit reticulocyte lysate . Two polypeptides corresponding to the N-terminal and C-terminal regions of the viral polyprotein were expressed in Escherichia coli and used to produce murine antisera for detection of translation products . Three major translation products of 113, 48, and 41 kDa were identified in a coupled transcription-translation system . The large 113-kDa protein reacted with antisera raised against the C-terminal region of the polyprotein and represents a precursor of the viral RNA polymerase . The 48-kDa protein detected in vitro reacted specifically with antisera raised against the polyprotein N terminus, showing that translation was initiated in SV at the three tandem in-frame AUG codons at the 5' end of the genome . A series of nested 3' deletions of the large open reading frame encoding the viral polyprotein was used to define the translation initiation site and genomic location of the viral protease . The results are consistent with a model in which translation of the viral genome is initiated at one of the three in-frame AUG codons starting at nucleotide position 5 and in which active viral protease is produced following translation of a region located between NheI (nucleotide 3052) and SphI (nucleotide 4056), resulting in rapid cleavage of a large precursor protein . Abolition of the viral 3C-like protease activity by site-directed mutagenesis of the putative active-site cysteine (Cys-1238) resulted in production of a large protein of approximately 200 kDa which reacted with both N-terminal and C-terminal antisera . Two potential polyprotein cleavage sites containing the preferred picornaviral QG recognition site were identified on either side of the putative 2C-like helicase region of the polyprotein . Proteolysis at these positions would give rise to products with relative molecular masses identical to those of the products detected in the rabbit reticulocyte system . Site-directed mutagenesis was used to introduce a single base change which resulted in the substitution of glutamine residues with proline residues at amino acids 399 and 762 . These mutations completely abolished cleavage of the polyprotein at these positions and gave rise to alternative products with molecular masses which matched the predicted sizes for a single cleavage at either Q-399 or Q-762 . These data indicate that the small, round-structured virus Southampton virus produces a 3C-like protease which has two primary cleavage sites at positions 399 and 762 . Proteolytic cleavage at these positions releases the putative viral 2C-like helicase.

J Virol, 1996 Apr, 70(4), 2497 - 502
Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer; Liu ML et al.; Recombinant retrovirus vectors are widely used for gene transfer studies . The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo . A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector . Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector . High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer . Subsequent studies demonstrated that beta-galactosidase protein was concentrated and copurified during pseudotype vector preparation, resulting in high rates of protein transfer rather than stable gene transfer, a process referred to as pseudotransduction . The cotransfer of protein with concentrated pseudotyped retroviruses indicates that caution must be used when interpreting gene transduction efficiencies in gene therapy experiments.

J Mol Evol, 1996 Apr, 42(4), 414 - 21
The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae; Rouhbakhsh D et al.; The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R . maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units . Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway . The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host . The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication . Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome . The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA) . In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II) . Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages . Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids.

Arch Biochem Biophys, 1996 Apr 1, 328(1), 78 - 84
Stability and folding of precursor and mature tryptophan-substituted ribose binding protein of Escherichia coli; Lee H et al.; A mutant ribose binding protein (RBP) of Escherichia coli was obtained by site-directed mutagenesis, replacing Phe-187 in the wild-type RBP (WT-RBP) with a Trp residue, in order to compare its stability and folding behavior with those of the WT-RBP . The equilibrium unfolding properties and the folding kinetics of these proteins were monitored by fluorescence and circular dichroism (CD) . For both WT-RBP and the Trp-substituted RBP (Trp-RBP), the conformational stabilities of the precursor proteins and the mature proteins were the same, indicating that the signal peptide had no influence on the property of the mature domain . The Phe/Trp substitution in the mature domain, however, brought about a significant decrease in the conformational stability . The signal peptide had an appreciable retarding effect on the folding of the precursor Trp-RBP as was reported for the WT-RBP . Refolding kinetics of the WT-RBP and Trp-RBP showed a two-step reaction when monitored by fluorescence and by CD.

Arch Biochem Biophys, 1996 Apr 1, 328(1), 173 - 83
Structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases; Edwards KJ et al.; Quinone oxidoreductase, zeta-crystallin, glucose dehydrogenase, and alcohol dehydrogenase belong to a superfamily of medium-chain dehydrogenase/reductases . The crystal structures of Escherichia coli quinone oxidoreductase (QOR) and Thermoplasma acidophilum glucose dehydrogenase have recently been determined and are compared here with the well-known structure of horse liver alcohol dehydrogenase . A structurally based comparison of these three enzymes confirms that they possess extensive overall structural homology despite low sequence identity . The most significant difference is the absence of the catalytic and structural zinc ions in QOR . A multiple structure-based sequence alignment has been constructed for the three enzymes and extended to include zeta-crystallin, an eye lens structural protein with quinone oxidoreductase activity and high sequence identity to E . coli quinone oxidoreductase . Residues which are important for catalysis have been altered and the functions and activities of the enzymes have diverged, illustrating a classic example of divergent evolution among a superfamily of enzymes.

Arch Biochem Biophys, 1996 Apr 1, 328(1), 107 - 14
The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates; Hook VY et al.; Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes . The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes . The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates . The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies . Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones . PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC . In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY . PC1/3 and PC2 preferred POMC among the prohormones tested . Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.

J Clin Endocrinol Metab, 1996 Apr, 81(4), 1389 - 96
Recombinant synthesis of insulin-like growth factor-binding protein-4 (IGFBP-4): Development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids; Honda Y et al.; Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological effects . To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP-4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione-derivatized resin . Antibody against the rhIGFBP-4 fusion protein was raised in guinea pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography . We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunoreactive with the IGFBP-4 antiserum . Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample . We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level . Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L) . The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01) . In addition, serum IGFBP-4 amounts showed a significant positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01) . These data validate this IGFBP-4 RIA and illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging.

J Bacteriol, 1996 Apr, 178(8), 2469 - 70
The tyrT locus of Escherichia coli B; Timms AR et al.; The tyrT (tRNA(1TYr)) locus of Escherichia coli B differs structurally from that of K-12 strains by the absence of 2 of 3.14 terminal repeat sequences.

J Bacteriol, 1996 Apr, 178(8), 2445 - 9
Isolation of a pdxJ point mutation that bypasses the requirement for the PdxH oxidase in pyridoxal 5' -phosphate coenzyme biosynthesis in Escherichia coli K-12; Man TK et al.; We isolated 26 suppressor mutations that allowed growth of a delta pdxH::omega null mutant in the absence of pyridoxal . Each suppressor mapped to pdxJ, and the eight suppressors sequenced contained the same glycine-to-serine change in the PdxJ polypeptide . This bypass suppression suggests that PdxJ may participate in formation of the pyridine ring of pyridoxine 5'-phosphate.

J Bacteriol, 1996 Apr, 178(8), 2436 - 9
Analysis of the spacer DNA between the cyclic AMP receptor protein binding site and the lac promoter; Flatow U et al.; The role of the spacer region DNA between the cyclic AMP receptor protein (CRP) site and the RNA polymerase in the lac promoter was examined . We wanted to determine whether the wild-type DNA sequence of this region was an absolute requirement for CRP activation of lac transcription . The sequence of a 9-bp stretch of the spacer, from -41 to -49 relative to the start of transcription, was randomized, and the effect of randomization on lac expression was investigated in vitro and in vivo . We found that the spacer contains no specific sequence determinants for CRP activation of lac transcription; fewer than 1% of the mutants displayed greater than a 50% decrease in CRP activation of lac transcription.

J Bacteriol, 1996 Apr, 178(8), 2420 - 3
The insE open reading frame of IS1 is not required for formation of cointegrates; Freund ET et al.; The role of the insE open reading frame in transposition of IS1 was reexamined by using an insE nonsense mutation that does not alter the amino acid sequence of InsA inhibitor or InsAB transposase . The mutant was active in all strains tested, showing that insE is not essential for formation of cointegrates.

J Bacteriol, 1996 Apr, 178(8), 2388 - 96
Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells; Feng G et al.; The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli . We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting . Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards . In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell . Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells . Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used . In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria . RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells . These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH . Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis . On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single-stranded DNA encoded by bacterial retrons.

J Bacteriol, 1996 Apr, 178(8), 2383 - 7
A hairpin structure upstream of the terminator hairpin required for ribosomal protein L4-mediated attenuation control of the S10 operon of Escherichia coli; Zengel JM et al.; Ribosomal protein L4 of Escherichia coli regulates transcription of the 11-gene S1O operon by promoting premature termination of transcription (attenuation) at a specific site within the 172-base untranslated leader . We have analyzed the roles of various domains of the leader RNA in this transcription control . Our results indicate that the first 60 bases of the leader, forming the three proximal hairpin structures, are not essential for in vivo L4-mediated attenuation control . However, a deletion removing the fourth hairpin, which is immediately upstream of the terminator hairpin, eliminates L4's effect on transcription . Base changes disrupting complementarity in the 6-bp stem of this hairpin also abolish L4 control, but compensatory base changes that restore complementarity also restore L4's effect . In vitro transcription studies confirm that this hairpin structure is necessary for L4's role in stimulating transcription termination by RNA polymerase.

J Bacteriol, 1996 Apr, 178(8), 2362 - 7
Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot; Ukita T et al.; Illegitimate recombination between a prophage and adjacent bacterial DNA is the first step in the formation of specialized transducing phage . Such recombination is rare, but it is greatly enhanced by UV irradiation . We studied the mechanism of UV-induced illegitimate recombination by examining the effect of rec mutations on the frequency of lambda bio transducing phage and found that an Escherichia coli recJ mutation reduces it by 3- to 10-fold . In addition, the recombination hotspot, which accounts for approximately 60% of lambda bio transducing phages in wild-type bacteria, was not detected in the recJ mutant . Introduction of a RecJ overexpression plasmid into the recJ mutant recovered the recombination at the hotspot . These results indicate that the RecJ protein preferentially stimulates illegitimate recombination at the hotspot . Both the hotspot and the non- hotspot sites have short regions of homology, but only the hotspot sites contain common direct-repeat sequences . We propose a model based on the 5'-3' exonuclease activity of RecJ to explain the involvement of this protein in illegitimate recombination at the hotspot.

J Bacteriol, 1996 Apr, 178(8), 2255 - 62
Characterization of transmembrane domains 6, 7, and 8 of MalF by mutational analysis; Ehrle R et al.; Oligonucleotide mutagenesis was used to isolate mutations in membrane-spanning segments 6, 7, and 8 of MalF . MalF is a cytoplasmic membrane component of the binding protein-dependent maltose transport system in Escherichia coli . The current structural model predicts eight transmembrane domains for MalF . Membrane-spanning segments 6, 7, and 8 of MalF flank or are part of the EAA-X3-G-X9-I-X-LP consensus region present in the cytoplasmic membrane subunits of the bacterial ABC transporter superfamily members . Mutations with two novel phenotypes with respect to substrate specificity of the maltose transport system were isolated . One mutant grew on minimal maltose media but not on media containing either maltoheptaose or maltoheptaose plus maltose and was thus termed dextrin dominant negative . The other class of mutations led to a maltose minus but maltoheptaose plus phenotype . Nine of the isolated mutations leading to changes in substrate specificity were tightly clustered on one face of the postulated transmembrane helix 6 . A similar clustering of mutations was detected in transmembrane domain 7 . The majority of mutations in membrane-spanning segment 7 led to a protease-sensitive or a conditional phenotype with respect to MalF function or both . Mutations in transmembrane domain 8 appeared to be more randomly distributed . The majority of mutations in membrane-spanning segment 8 caused a Mal+ Dex- phenotype . Six Mal+ suppressor mutations isolated to two mutations in transmembrane domain 7 changed amino acid residues in membrane-spanning segment 6 or 8.

J Bacteriol, 1996 Apr, 178(8), 2211 - 5
Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides; Markaryan A et al.; The function of the long propeptides of fungal proteinases is not known . Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family . These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides . Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography . A . fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A . flavus was less strongly inhibited and subtilisin was not inhibited . Binding of metalloproteinase propeptide from A . fumigatus to the mature metalloenzyme was demonstrated . This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A . flavus were not inhibited by this propeptide . Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment . These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion.

J Bacteriol, 1996 Apr, 178(8), 2172 - 7
Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa; Luo Z et al.; The Neurospora crassa arg-2 transcript contains an upstream open reading frame (uORF) specifying a 24-residue leader peptide and is subject to a novel form of negative translational regulation in response to arginine . The role of the arg-2 uORF in arginine-specific negative regulation was investigated by using translational fusions of wild-type and mutant arg-2 sequences to the Escherichia coli lacZ reporter gene specifying beta-galactosidase . The wild-type uORF conferred Arg-specific regulation on the reporter gene in N . crassa, but mutated or truncated uORFs did not, as determined by measurements of beta-galactosidase activity produced in N . crassa strains expressing arg-2-lacZ fusion genes . All effects on reporter gene expression were posttranscriptional, as determined by measurement of RNA levels . Both sequence-dependent and sequence-independent effects of uORFs were observed . Genes containing the wild-type uORF or a 21-codon mutated uORF showed reduced translation in comparison with that of a gene lacking a uORF . Both uORF-containing transcripts showed reduced association with polysomes relative to transcripts lacking a uORF, but only the transcript with the wild-type uORF showed a reduced average number of ribosomes associated with it in response to arginine addition . Direct translational fusions between uORF sequences and lacZ sequences indicated that the uORF is translated . Overlapping the uORF with the lacZ initiation codon indicated that ribosome reinitiation at a downstream start codon is not integral to uORF-mediated, Arg-specific translational regulation . These studies provide direct biochemical evidence for arg-2 uORF function in translational control.

Circ Res, 1996 Apr, 78(4), 547 - 52
Vital staining of cardiac myocytes during embryonic stem cell cardiogenesis in vitro; Metzger JM et al.; Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes . The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures . The -440 to +6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro . Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418 . Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro . Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity . The earliest detection was at day 6 of differentiation, when approximately 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximately 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes . Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture . In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro . The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.

RNA, 1996 Apr, 2(4), 354 - 66
Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB; Huttenhofer A et al.; On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling . We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions . The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region . Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis . The second domain is located within the apical loop of the mRNA hairpin structures . This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions . The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix . Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21) . The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix . Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs . Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures . Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM.

RNA, 1996 Apr, 2(4), 299 - 307
Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release; Tallsjo A et al.; We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P . Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection . The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA . Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA . Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release . In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives . These precursors carry a C at the discriminator base position . The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site . The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA . This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection.

Int Arch Allergy Immunol, 1996 Apr, 109(4), 356 - 61
Direct expression of Der f2, a major house dust mite allergen, in Escherichia coli; Iwamoto N et al.; Der f2 protein in a highly antigenic form was directly expressed in bacteria . Plasmid pFLU11 derived from pKK233-2 was designed to express methionyl-Der f2 under the control of the trc promoter and it has the replication origin of pUC118 instead of its original to increase the copy number . This expression plasmid directed the synthesis of recombinant Der f2 (rDer f2) protein in an insoluble form of inclusion bodies in Escherichia coli cells . The high copy number plasmid pFLU11 conferred the efficient production of the Der f2 protein in E . coli, when compared to a nonchanged origin material . rDer f2 inclusion bodies were easily solubilized in urea and renatured by dialysis to assume the active form . The rDer f2 protein was purified by means of anion exchange and gel filtration chromatography . This expression system yielded about 10 mg of purified rDer f2 protein from the 1L culture . Purified rDer f2 protein reacted with IgE from patient sera almost identically to the native Der f2 in the RAST enzyme immunoassay and skin prick test.

J Gen Virol, 1996 Apr, 77 ( Pt 4), 587 - 92
Cooperative binding to nucleic acids by barley yellow mosaic bymovirus coat protein and characterization of a nucleic acid-binding domain; Reichel C et al.; The capacity of several coat protein (CP) mutants of a German isolate of barley yellow mosaic bymovirus (BaYMV) to bind of nucleic acids was studied in vitro . Recombinant CP, produced by overexpression in Escherichia coli, was purified from inclusion bodies and subsequently renatured . Binding to single-stranded (ss) RNA and ssDNA oligonucleotides was found to be cooperative and sequence non-specific . By deletion mutagenesis, several truncated CP derivatives were created and their nucleic acid-binding capacity was investigated in order to define a protein domain responsible for RNA- and DNA-binding . The nucleic acid-binding domain consists of a core which was located to an internal 23 amino acid peptide (aa 125-147) and an adjacent domain (aa 148-184) which stimulates binding.

J Gen Virol, 1996 Apr, 77 ( Pt 4), 567 - 73
Purification, characterization, assembly and crystallization of assembled alfalfa mosaic virus coat protein expressed in Escherichia coli; Yusibov V et al.; The coast protein of alfalfa mosaic virus (AMV) was cloned and expressed in Escherichia coli as a fusion protein containing a 37 amino acid extension with a (His)6 region for affinity purification . About half of the expressed recombinant coat protein (rCP) was soluble upon extraction and half was insoluble in inclusion bodies . Western blot analysis confirmed the identity of the rCP and protoplast infectivity assays indicated that the rCP was biologically active in an early event of AMV infection, called genome activation . The rCP assembled into T = 1 empty icosahedral particles, as described previously for native coat protein . Empty particles formed hexagonal crystals that diffracted X-rays to 5.5 A resolution . The crystals of trypsin-treated particles of rCP appear to be isomorphous with crystals of trypsin-treated particles of native coat protein, Spherical particles containing RNA assembled when the rCP was combined with in vitro transcripts of AMV RNA4, the smallest naturally encapsidated AMV RNA . Bacilliform particles that resembled native virions assembled when the rCP was combined with transcripts of RNA1, the largest genomic RNA.

Int J Radiat Biol, 1996 Apr, 69(4), 459 - 69
Heavy ion inactivation in Escherichia coli cells: experiment and theory; Schafer M; Cross sections for inactivation of stationary cells of E . coli strains (B(s-1) and B/r) by heavy ions (Z = 8-92) with energies <15 MeV/u were determined from survival curves and compared with track structure calculations . Whereas for low energies, inactivation cross sections for both strains are similar, and thus do not reflect their difference in X-ray sensitivity, the ratios of cross sections of the two strains approach the ratio of X-rays sensitivities for heavy ions at high energies . The essential features of the experimental data are reproduced well by the track structure calculations, which do not use fitted values for the model parameters but independently determined values for the X-ray sensitivities and the size of the target structure . Quantitatively, the calculations underestimate the cross sections for heavier ions as well as their ratios at higher energies . Some possible reasons for these discrepancies are discussed.

Eur J Immunol, 1996 Apr, 26(4), 886 - 91
Regions of RAG1 protein critical for V(D)J recombination; Kirch SA et al.; The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors . To investigate the function of RAG1, we have tested a series of insertion and substitution mutation for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates . With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly . As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conversed sequence . We show here that this "dispensable" region of RAG1 is not necessary for coding joint formation or recombination of an integrated substrate, and this portion is not functionally redundant with the "dispensable" region of RAG2 . Recombination with these core regions is also still subject to the 12/23 joining rule . Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.

Carcinogenesis, 1996 Apr, 17(4), 809 - 13
Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by human cytochrome P450 1A2 and its inhibition by phenethyl isothiocyanate; Smith TJ et al.; 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific nitrosamine in animals and has been suggested to play a role in human tobacco-related cancers . Our previous study demonstrated that cytochrome P450 (P450) 1A2 catalyzes the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol) (an alpha-hydroxylation product) from NNK in human liver microsomes . Phenethyl isothiocyanate (PEITC) inhibits NNK tumorigenesis by blocking the activation of NNK . The purpose of the present study was to elucidate the mechanism of inhibition of P450 1A2-catalyzed NNK activation by PEITC . Human P450 1A2 was expressed in Escherichia coli and purified to homogeneity . In a reconstituted system, P450 1A2 catalyzed the formation of keto alcohol and 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) from NNK, with the keto alcohol being the major metabolite . The apparent Km and Vmax values for keto alcohol formation was 380 microM and 1.7 nmol/min/nmol P450, respectively . For the tobacco-specific nitrosamine N-nitrosonornicotine (NNN), P450 1A2 catalyzed the formation of the derived 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid),4-oxo-4-(3-pyridyl)butyric acid (keto acid) and keto alcohol . In comparison to NNK, NNN had a lower rate of oxidation with P450 1A2 . PEITC decreased the formation of the NNK-derived keto alcohol in a concentration-dependent manner, with an IC50 value of 0.14 microM . PEITC was a competitive inhibitor of P450 1A2, exhibiting a Ki value of 0.18 microM . Preincubation of PEITC with NADPH in the reconstituted system resulted in a further decrease (25%) in the catalytic activity of P450 1A2, suggesting that there is a slow metabolism-dependent inhibition of P450 1A2 by PEITC . The formation of keto aldehyde and keto alcohol was inhibited by PEITC in human liver microsomes with IC50 values of 9.5 and 4.6 microM respectively . Methoxyresorufin O-dealkylase activity, a marker for P450 1A2, was decreased by PEITC in a concentration-dependent manner, with an IC50 of 0.34 microM . The results suggest that PEITC itself is a potent inhibitor of P450 1A2 and that a metabolite(s) of PEITC can also inhibit P450 1A2 . We conclude that PEITC may be an effective inhibitor of the carcinogenicity or toxicity of chemicals that are activated by P450 1A2.

Curr Genet, 1996 Apr, 29(5), 482 - 9
Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum; Fierro F et al.; Plasmid vectors containing the AMA1 sequence transformed with high efficiency and replicated autonomously in Penicillium chrysogenum . The efficiency of transformation of P . chrysogenum was related to the length of the AMA1 fragment used for constructing the different autonomously replicating plasmids . One of the two palindromic inverted repeats of AMA1 (the 2.2-kb SalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency . Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number . Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency . Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication . The AMA1 region works as an origin of replication in P . chrysogenum and A . nidulans but not apparently in Acremonium chrysogenum.

Curr Genet, 1996 Apr, 29(5), 446 - 56
The 3-phosphoglycerate kinase gene of the yeast Yarrowia lipolytica de-represses on gluconeogenic substrates; Le Dall M et al.; We have isolated the 3-phosphoglycerate kinase (PGK) gene of the yeast Yarrowia lipolytica by probing a genomic library with a PCR fragment amplified with primers deduced from two highly conserved regions of various PGKs . It is a unique sequence encoding a polypeptide of 417 residues with extensive homology to other PGKs, especially to that of Aspergillus nidulans (76% identity) . The expression of the Y . lipolytica PGK1 gene proved to be higher on gluconeogenic substrates than on glycolytic ones . Haploid strains harboring a disrupted allele were able to grow on mixtures of a gluconeogenic carbon source and of a glycolytic one, but required proline supplementation in the presence of glucose, and were inhibited by glycerol.

Eur J Biochem, 1996 Apr 1, 237(1), 58 - 63
Structural and functional characterization of two mutated R2 proteins of Escherichia coli ribonucleotide reductase; Larsson A et al.; The R2 protein of ribonucleotide reductase from Escherichia coli is a homodimeric tyrosyl-radical-containing enzyme with two identical dinuclear iron centers . Two randomly generated genomic mutants, nrdB-1 and nrdB-2, that produce R2 enzymes with low enzymatic activity, have been cloned and characterized to identify functionally important residues and areas of the enzyme . The mutations were identified as Pro348 to leucine in nrdB-1 and Leu304 to phenylalanine in nrdB-2 . Both mutations are the results of single amino acid replacements of non-conserved residues . The three-dimensional structures of {L348}R2 and {F304}R2 have been determined to 0.26-nm and 0.28-nm resolution, respectively . Compared with wild-type R2, {L348}R2 binds with higher affinity to R1, probably due to increased flexibility of its C-terminus . Since the three-dimensional structure, iron-center properties and radical properties of {L348}R2 are comparable to those of wild-type R2, the low catalytic activity of the holoenzyme is probably caused by a perturbed interaction between R2 and R1 . The {F304}R2 enzyme has increased radical sensitivity and low catalytic activity compared with wild-type R2 . In {F304}R2 the only significant change in structure is that the evolutionary conserved Ser211 forms a different hydrogen bond to a distorted helix . The results obtained with {F304}R2 indicate that structural changes in E . coli R2 in the vicinity of this helix distortion can influence the catalytic activity of the holoenzyme.

Eur J Biochem, 1996 Apr 1, 237(1), 318 - 21
Potassium ions and the molecular-chaperone activity of DnaK; Feifel B et al.; Potassium ions stabilize the DnaK.ADP complex that forms on incubation of nucleotide-free DnaK with ADP or ATP . Generation of the crystallographically defined Mg2+ cluster {Wilbanks, S.M . & McKay, D.B . (1995) J . Biol . Chem . 270, 2251-2257}, in which two K+ and the nucleotide are bound together with Mg2+ in the ATPase site, appears to be essential for the ATP-induced acceleration of binding of peptide ligands, the ATP-induced release of peptide ligands and for the peptide-induced increase in ATPase activity . Thus, K+ is instrumental in signal transmission between the ATPase site and the peptide-binding site.

Eur J Biochem, 1996 Apr 1, 237(1), 295 - 302
Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity; Ruppert R et al.; Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development . A cDNA encoding mature human BMP-2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP-2 protein of M(r) 26,000 was prepared with a purity greater 98% . The recombinant BMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC50 of 15-20 nM) . A peptide 1-17 representing the N-terminal basic part of BMP-2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay . These observations suggested that the N-terminai reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracellular matrix . This conclusion was supported by a variant EHBMP-2, where the N-terminal residues 1-12 of BMP-2 had been substituted by a dummy sequence of equal length and which showed an EC50 value of around 1 nM which was affected neither by heparin nor by peptide 1-17 . A physical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin-binding of variant EHBMP-2 was negligible . These results identify the basic N-terminal domains of dimeric BMP-2 as heparin-binding sites that are not obligatory for receptor activation but modulate its biological activity.

Eur J Biochem, 1996 Apr 1, 237(1), 247 - 54
Escherichia coli isocitrate dehydrogenase kinase/phosphatase . Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues; Rittinger K et al.; The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus . By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity . The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm . The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein . Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide . The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, were used to investigate the interaction with IDH K/P . The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides . In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides . The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C . Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP.

Eur J Biochem, 1996 Apr 1, 237(1), 143 - 52
Translational control of poly(A)-binding protein expression; Bag J et al.; Poly(A)-binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract . In many eukaryotic cells, this mRNA is inefficiently translated . The 5' untranslated region (UTR) of PABP mRNA has several adenine-rich regions which may serve as the PABP-binding sites to control its translation by a feed-back mechanism . This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell-free system . Results of our studies show that removal of the putative PABP-binding sites from the 5' UTR of this mRNA enhances its translation in the rabbit reticulocyte cell-free system . Furthermore, in vitro translation of the full-length PABP mRNA was inhibited by addition of purified PABP to the cell-free system . In contrast, translation of truncated mRNA lacking the putative PABP-binding sites at the 5' UTR was not inhibited by exogenous PABP . We have also tested the ability of purified PABP to bind to the 5' UTR of PABP mRNA using ultraviolet-mediated covalent cross-linking of RNA and proteins in vitro . Our results show that exogenous PABP binds to the 5' UTR of its full-length mRNA . Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5' UTR . The adenine-rich regions are located within this segment of PABP mRNA . Following incubation of PABP mRNA in the reticulocyte lysate cell-free system under conditions of mRNA translation, the polysomal and non-translated free mRNA fractions were separated by centrifugation . Analysis of free and polysomal mRNA-protein (mRNP) complexes following ultraviolet-induced cross-linking showed that the free mRNP population was preferentially enriched in PABP . Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed-back mechanism.

Eur J Biochem, 1996 Apr 1, 237(1), 128 - 35
Comparison of the structural features of ubiquinone reduction sites between glucose dehydrogenase in Escherichia coli and bovine heart mitochondrial complex I; Sakamoto K et al.; To characterize the structural features of the ubiquinone reduction site of glucose dehydrogenase (GlcDH) in Escherichia coli, we performed structure/activity studies of a systematic set of synthetic ubiquinone analogues and specific inhibitors (synthetic capsaicins) of this site . Considering the proposed similarity of the quinone binding domain motif between GlcDH and one subunit of mitochondrial complex I {Friedrich, T., Strohdeicher, M., Hofhaus, G., Preis, D., Sahm, H . & Weiss, H . (1990) FEBS Lett . 265, 37-40}, we compared the structure/activity profiles of the substrates and inhibitors for GlcDH with those for bovine heart mitochondrial complex i . With respect to GlcDH, replacement of one or both methoxy groups in the 2 and 3 positions of benzoquinone ring by ethoxy group(s) resulted in a drastic decrease in the electron accepting activity . The presence of a 5-methyl group and the conformational property of the 6-alkyl side chain did not significantly contribute to the activity . These results suggested that only half of the benzoquinone ring (the moiety corresponding to the 2 and 3 positions) is recognized by the quinone reduction site in a strict sense . In contrast, quinone analogues with structural modifications at all positions in the benzoquinone ring retained the activity with mitochondrial complex I . This finding indicated that the catalytic site of complex I is spacious enough to accommodate a variety of structurally different quinone derivatives . The correlation of the inhibitory potencies of a series of synthetic capsaicins between the two enzymes was very poor . These findings indicated that the binding environment of ubiquinone in GlcDH is very specific and differs from that in mitochondrial complex I.

Anesth Analg, 1996 Apr, 82(4), 810 - 6
Mild intraoperative hypothermia reduces production of reactive oxygen intermediates by polymorphonuclear leukocytes; Wenisch C et al.; Mild hypothermia directly impairs numerous immune functions in vitro . However, the in vivo effects of mild hypothermia on neutrophil phagocytosis and oxidative killing remain unknown . We tested the hypothesis that mild intraoperative hypothermia decreases neutrophil phagocytic capacity and generation of reactive oxygen intermediates (a measure of oxidative killing) . Additionally, we evaluated the effects of in vitro temperature manipulations on each function . Thermal management was randomly assigned in 10 surgical patients, causing intraoperative core temperatures to range from 33 to 37 degrees C . Production of reactive oxygen intermediates and neutrophil phagocytosis were evaluated using flow cytometry at ambient temperature . Phagocytic capacity was assessed by uptake of fluorescein isothiocyanate-labeled Escherichia coli . Reactive oxygen production was estimated by the intracellular conversion of dihydrorhodamine 123 to rhodamine 123 . Blood samples were obtained preoperatively, 1 h after surgery started, and 2 h postoperatively . Blood was also obtained from 10 matched control subjects and tested at 32, 37, and 40 degrees C . Neutrophil oxidative and phagocytic capacities were significantly reduced intraoperatively, compared with preoperative and postoperative values . Intraoperative production of reactive oxygen species was linearly related to core temperature . In contrast, there was no correlation between core temperature and phagocytic activity . In vitro production of reactive oxygen intermediates increased sixfold from 32 to 40 degrees C . In vitro phagocytic capacity increased fourfold in this temperature range . Production of oxidative intermediates was most closely related to intraoperative core temperature, decreasing nearly fourfold over a 4 degree C range . This in vitro temperature dependence was matched in vitro . Impaired neutrophil oxidative killing may contribute to the observed hypothermia-induced reduction in resistance to infection.

Virology, 1996 Apr 1, 218(1), 1 - 13
Mutations in the poliovirus 3CD proteinase S1-specificity pocket affect substrate recognition and RNA binding; Blair WS et al.; Sequence and structure comparisons with homologous trypsin-like serine proteases have predicted the S1-specificity pocket in picornavirus 3C proteinases . In this study, we examine the putative roles of such residues in poliovirus 3C substrate recognition . Single amino acid substitutions at 3C residues Thr-142, His-161, Gly-163, Gly-164, and Ala-172 were introduced into near full-length poliovirus cDNAs, and protein processing was examined in the context of authentic 3C cis cleavage activity . Our data are consistent with residues Thr-142, His-161, Gly-163, and Gly-164 acting as important determinants of 3C substrate specificity and support published models of 3C protein structure . An in vivo analysis of mutant viruses containing individual amino acid substitutions at 3C residues Thr-142 and Ala-172 suggests that such residues are important determinants for viral RNA replication . In addition, bacterially expressed, recombinant 3CD polypeptides containing amino acid substitutions at Thr-142 and Ala-172 show altered RNA binding properties in mobility shift assays that use a synthetic RNA corresponding to the poliovirus 5'-terminal sequences.

Nucleic Acids Res, 1996 Apr 1, 24(7), 1380 - 1
Kinetics of transcription in a minute column; Kubori T et al.; Immobilized enzymes or nucleic acids are widely used to analyze association and dissociation reactions . A pseudo-rapid kinetic method for studying transcription was developed by using an immobilized template DNA packed in a minute column, in a micro-scale liquid chromatography system . This method, which has a dead-time of only several seconds, is quick enough to allow analysis of the release of product RNA in transcription by Escherichia coli RNA polymerase . The method could be applied to most enzymes that interact with DNA.

Nucleic Acids Res, 1996 Apr 1, 24(7), 1308 - 13
Differential discrimination of DNA polymerase for variants of the non-standard nucleobase pair between xanthosine and 2,4-diaminopyrimidine, two components of an expanded genetic alphabet; Lutz MJ et al.; Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) were examined for their ability to incorporate components of an expanded genetic alphabet in different forms . Experiments were performed with templates containing 2'-deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanthosine (c7dX), both able to adopt a hydrogen bonding acceptor-donor-acceptor pattern on a purine nucleus (puADA) . Thus these heterocycles are able to form a non-standard nucleobase pair with 2,4-diaminopyrimidine (pyDAD) that fits the Watson-Crick geometry, but is joined by a non-standard hydrogen bonding pattern . HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH . Specific incorporation opposite c7dX was significantly lower and also independent of pH . Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E . coli DNA polymerase I failed to incorporate d(pyDAD)TP opposite c7dX.

Nucleic Acids Res, 1996 Apr 1, 24(7), 1279 - 86
Incomplete factorial and response surface methods in experimental design: yield optimization of tRNA(Trp) from in vitro T7 RNA polymerase transcription; Yin Y et al.; We have studied the yield of Escherichia coli tRNA(Trp) obtained from in vitro T7 RNA polymerase transcription using incomplete factorial and response surface methods . Incomplete factorial experiments were first used to estimate the relative impact of six variables on the yield of tRNA(Trp) . Fifteen trials were performed according to a balanced and randomized design . The correlation between observed yield and all experimental variables was identified by stepwise multiple linear regression analysis . The concentrations of T7 RNA polymerase, DNA template, NTP and MgCl2 proved to be significantly correlated with the yield of tRNA(Trp) . We then optimized the yield with respect to each of these four variables simultaneously with a designed, response surface experiment based on the Hardin-Sloane minimum prediction variance algorithm . Twenty experiments were performed, in duplicate, to sample the quadratic surface relating the yield to the four significant variables . Coefficients of the quadratic function with all two-factor interactions were evaluated by stepwise regression using least squares, and significant coefficients were retained . Partial differentiation of the resulting quadratic model showed it to possess an optimum . Transcription performed at the corresponding conditions yielded 6-fold more tRNA(Trp) than the initial conditions, confirming the predictive value of the experimentally determined response surface.

Nucleic Acids Res, 1996 Apr 1, 24(7), 1179 - 86
A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands; Eggleston AK et al.; We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity . This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding . These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding . We evaluated the potential of several dyes {acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange} to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme . Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes . The effects of temperature and salt concentration on the rate of unwinding were also examined . We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.

J Trauma, 1996 Apr, 40(4), 557 - 62; discussion 563
Prostaglandin E2 production by endotoxin-stimulated alveolar macrophages is regulated by phospholipase C pathways; Lo CJ et al.; BACKGROUND: Eicosanoids play an important role in many aspects of systemic inflammatory responses and host defense . Although the synthesis of eicosanoids by different enzymes has been elucidated, the regulatory mechanism of eicosanoid production is not clear . We designed this study to investigate the hypothesis that PGE2 production by endotoxin (lipopolysaccharide; LPS)-stimulated macrophages (MO) is dependent on phospholipase C (PLC) signaling pathways . METHODS: Rabbit alveolar macrophages (MO) were obtained by bronchoalveolar lavage . MO were suspended in RPMI-1640 medium at 1 x 10(6)/mL and were exposed to Escherichia coli LPS (10 ng/mL) +/- various agonists and antagonists of PLC and its secondary messengers . After 24 hours of incubation, prostaglandin E2 (PGE2) production was measured by ELISA . RESULTS: LPS-activated MO produced four times as much PGE2 as did control unstimulated MO . The increase in PGE2 production was inhibited by PLC inhibitors (U73122 or D609) and a low-molecular-weight PLA2 inhibitor, manoalide . An increase in intracellular calcium and activation of both the calmodulin and protein kinase C kinase pathways increase PGE2 production . CONCLUSIONS: PGE2 production is intimately dependent on several phospholipases . Production is not only dependent on low-molecular-weight PLA2 cleavage of arachidonic acid from membrane phospholipids, but also by-products of PLC activation . PLC-dependent intracellular Ca-calmodulin signaling and protein kinase C activation provide significant modulation of PGE2 production.

J Pharmacol Exp Ther, 1996 Apr, 277(1), 299 - 307
A pharmacological analysis of receptor subtypes and the mechanisms mediating the biphasic response induced by kinins in the rat stomach fundus in vitro; Cabrini DA et al.; Bradykinin (BK), des-Arg9-BK (DABK) and related kinins caused biphasic response (BR) in rat stomach fundus (RSF) precontracted with BaCl2 . The B2 receptor antagonist HOE 140 (3-30 nM) produced parallel rightward shifts of the contractile concentration-response curve (CRC) for BK, yielding a pA(2) value of 9.07 +/- 0.27 and slope of 0.99, but caused only discrete rightward shift of the relaxant CRC for BK, leaving the BR CRC to DABK unaffected . The B1 receptor antagonist des-Arg9-NPC 17761 (10 nM to 1 microM) caused graded rightward shifts of the relaxant (but not the contractile) CRC to DABK, yielding a pA2 value of 8.35 +/- 0.05 and slope of 0.59, but had no effect on BK-induced BR . BK- and DABK- (100 nM) induced relaxation were almost suppressed by apamin (1 microM) or by nifedipine (1 nM), but were unaffected by nitric oxide synthase inhibitors, methylene blue, lipo and cyclooxygenase inhibitors, selective receptor antagonist for histamine (H1 and H2), nicotine, platelet activating factor, tachykinins (both NK1 and NK2, calcitonin gene-related peptide, vasoactive intestinal peptide and ganglion blocking agent . BK- and DABK-mediated relaxations were reduced in Ca2+ -free medium plus EGTA, although BK-mediated contraction was more resistant . Escherichia coli endotoxin treatment (10 microgram /rat), 24 hr before, potentiated DABK-induced relaxation, but not contraction, and reduced BK-mediated relaxation (P < .05) . It is concluded that RSF express both B1 and B2 receptors . BK-induced contraction involves activation of B2 receptors, although DABK-induced relaxation is mediated by B1 receptors . Both B1 and B2 receptors in RSF are constitutive, but LPS treatment caused induction of B1 and down-regulation of B2 receptors . Finally, kinin-mediated relaxation in RSF are coupled to activation of Ca2+ activated K+ channels, and rely on Ca2+ influx via L-type voltage-sensitive channels.

EMBO J, 1996 Apr 1, 15(7), 1742 - 50
The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures; Makhov AM et al.; The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifically to the HSV-1 oriS and oriL origins of replication, and is a DNA helicase and DNA-dependent NTPase . In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS . In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex . In the presence of 4 mM ATP, binding at oriS was enhanced 2-fold, DNA loops or stem-loops were extruded from the UL9 protein complex at oriS, and the DNA in them frequently appeared highly condensed into a tight rod . The stem-loops contained from a few hundred to over one thousand base pairs of DNA and in most, oriS was located at their apex, although in some, oriS was at a border . The DNA in the stem-loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound the stem-loops strongly . Thus the DNA strands in the stem-loops exist in a partially paired, partially single-stranded state presumably making them available for ICP8 binding in vivo . These observations provide direct evidence for an origin specific unwinding by the HSV-1 UL9 protein and for the formation of a relatively stable four-stranded DNA in this process.

EMBO J, 1996 Apr 1, 15(7), 1696 - 704
Structure of the C-terminal end of the nascent peptide influences translation termination; Bjornsson A et al.; The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli . No general effects are found which can be attributed directly to the mRNA sequences itself . Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA . For the penultimate amino acid (-2 location), charge and hydrophobicity are important . For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors . The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA) . The influence of the -1 and -2 amino acids is cooperative . Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination . Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination . This restriction is not found for poorly expressed genes.

EMBO J, 1996 Apr 1, 15(7), 1650 - 7
White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein; Ballario P et al.; The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway . We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation . The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle . The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain . We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay . Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

Crit Care Med, 1996 Apr, 24(4), 642 - 6
Lidocaine attenuates the hypotensive and inflammatory responses to endotoxemia in rabbits; Taniguchi T et al.; OBJECTIVE: To assess the effects of lidocaine on the hemodynamic and inflammatory responses to Escherichia coli endotoxemia in rabbits . DESIGN: Prospective, randomized, controlled experimental study . SETTING: University laboratory . SUBJECTS: Twenty-seven female Japanese rabbits, anesthetized with urethane and ventilated mechanically . INTERVENTIONS: Animals were randomly assigned to one of three groups: a) endotoxemic control group (n = 9), receiving intravenous Escherichia coli endotoxin (0.5 mg/kg bolus) via the mesenteric vein; b) laparotomy control group (n = 9), treated identically to the endotoxemic control group, except for substitution of 0.9% saline for endotoxin; and c) lidocaine-treated group (n = 9), treated identically to the endotoxemic controls and additionally, intravenous lidocaine (3 mg/kg bolus, followed by infusion at 2 mg/kg/hr) was administered immediately after endotoxin MEASUREMENTS AND MAIN RESULTS: We compared hemodynamics, blood gases, and microscopic findings of lung tissue obtained at necropsy in each group . Laparotomy alone had a minimal effect on the parameters and findings . Endotoxin injection decreased mean systolic arterial pressure from 135 +/- 6 (SD) to 95 +/- 25 mm Hg (p < .05) and increased the mean base deficit from -1.2 +/- 1.8 to -14.4 +/- 4.2 mmol/L (p < .05), and caused the infiltration of neutrophils into the lungs . Lidocaine administration abolished the hypotension and attenuated the increase the base deficit to -9.5 +/- 2.1 mmol/L (p < .05) and the cellular infiltration in comparison with endotoxemic controls . CONCLUSIONS: Lidocaine attenuated the hemodynamic and inflammatory responses to endotoxemia in rabbits . Findings suggest that lidocaine administration may prevent the development of hypotension and metabolic acidosis during endotoxemia.

J Cell Biol, 1996 Apr, 133(2), 359 - 69
The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential; Chitaev NA et al.; Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc) . This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro . We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs . Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes . To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E . coli, are exposed to molecules containing the "C-domains" of several cadherins . These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin . Three separate segments of plakoglobin containing various numbers of the so-called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule . The binding pattern of plakoglobin segments in vitro is compared with that in vivo . Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.

J Surg Res, 1996 Apr, 62(1), 53 - 8
Steroid therapy can modulate gut barrier function, host defense, and survival in thermally injured mice; Gianotti L et al.; Prednisone may be immunosuppressive and dehydroepiandrosterone may stimulate the immune response, but their effect on gut-origin sepsis caused by bacterial translocation has not been studied . Balb/c mice were treated orally with prednisone (1 or 10 mg/kg/day) or saline for 4 days before receiving gavage with 10 (10) 14 C-labeled Escherichia coli and a 20% thermal injury . Mice were transfused with allogeneic blood and given dehydroepiandrosterone (5 or 25 mg/kg/day) or vehicle subcutaneously for 4 days before bacterial gavage and thermal injury . Some groups in each experiment were observed 10 days for mortality and others were sacrificed 4 hr postburn to measure translocation and survival of translocated bacteria . Survival in prednisone treated animals was 25% (10 mg/kg/day) and 75% (1 mg/kg/day) versus 80% for controls . Following dehydroepiandrosterone administration, survival was 72% (25 mg/kg/day/group) and 30% (5 mg/kg/day/group) versus 16% for controls . High dose prednisone increased bacterial translocation to the intestinal wall and mesenteric lymph nodes and greatly impaired killing of translocated E . coli . In contrast, dehydroepiandrosterone (25 mg/kg) did not affect translocation but significantly improved bacterial killing . Prednisone and dehydroepiandrosterone exert opposite effects during gut-derived sepsis.

J Surg Res, 1996 Apr, 62(1), 11 - 6
Gut barrier failure in experimental obstructive jaundice; Reynolds JV et al.; Bacterial translocation from the gastrointestinal tract is central to current concepts of endogenous sepsis . Studies were designed to evaluate the potential relevance of translocation to the high incidence of infection in obstructive jaundice . Sprague-Dawley rats underwent laparotomy and division of the bile duct or sham ligation . In Study 1, rats were sacrificed after 24 hr, 1 week, and 3 weeks and the mesenteric lymph node complex, cecum, and blood were cultured and plasma endotoxin was measured . In Studies 2 and 3, sham-and bile duct-ligated rats were challenged after 1 week with operative trauma and intravenous endotoxin, respectively . Animals were sacrificed after a further 24 hr . No translocation was observed in sham-operated rats . Although colonization of the mesenteric lymph nodes was not seen in bile duct-ligated rats after 24 hr, this was evident in 75% of rats after 1 and 3 weeks . Surgical trauma and endotoxin produced bacterial translocation in 33 and 40%, respectively, of sham-operated animals; this was enhanced in bile duct-ligated rats to 75% (P < 0.01 vs shams) and 93% (P < 0.001 vs shams), respectively . Endotoxin resulted in positive blood cultures in 71% of jaundiced rats compared with none of the sham group injected with endotoxin (P < 0.001) . Biliary obstruction produces bacterial translocation and this process is enhanced by surgical trauma and endotoxin . The data support the thesis of gut barrier failure in jaundice and suggest that therapies targeted toward decreasing bacterial translocation may merit evaluation in the prophylaxis and treatment of infection in the jaundiced patient.

J Bacteriol, 1996 Apr, 178(7), 2086 - 93
Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2; Sobecky PA et al.; A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli . The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination . To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E . coli . It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division . However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone . An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E . coli . Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E . coli strains tested . The stabilization found is not due to resolution of multimers . The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E . coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature . The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions . In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population . Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing . The relative contribution of each system to stabilization depends on plasmid copy number and the particular E . coli host.

J Bacteriol, 1996 Apr, 178(7), 2051 - 9
glc locus of Escherichia coli: characterization of genes encoding the subunits of glycolate oxidase and the glc regulator protein; Pellicer MT et al.; The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity . Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB . The gene glcC may encode the glc regulator protein . Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities . The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins . The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of this enzyme . No effect on glycolate metabolism was detected by insertional mutation in glcG . Northern (RNA) blot experiments showed constitutive expression of glcC but induced expression for the structural genes and provided no evidence for a single polycistronic transcript.

J Bacteriol, 1996 Apr, 178(7), 2044 - 50
Exclusion of T4 phage by the hok/sok killer locus from plasmid R1; Pecota DC et al.; The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copy-number plasmid R1 . To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and lambda phage . Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined . The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by approximately 85% . Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min . These results further suggest that single cells exhibit altruistic behavior.

J Bacteriol, 1996 Apr, 178(7), 2018 - 24
Genetic characteristics of new recA mutants of Escherichia coli K-12; Alexseyev AA et al.; To search for functionally thermosensitive (FT) recA mutations, as well as mutations with differently affect RecA protein functions, seven new recA mutations in three different regions of the RecA protein structure proposed by Story et al . {R . M . Story, I . T . Weber, and T . A . Steitz, Nature (London) 355:318-325, 1992} were constructed . Additionally, the recA2283 allele responsible for the FT phenotype of the recA200 mutant was sequenced . Five single mutations (recA2277, recA2278, recA2283, recA2283E, and recA2284) and one double mutation (recA2278-5) generated, respectively, the amino acid substitutions L-277-->N, G-278-->P, L-283-->P, L-283-->E, I-284-->D, and G-278-->T plus V-275-->F in the alpha-helix H-beta-strand 9 region of the C-terminal domain of the RecA protein structure . According to recombination, repair, and SOS-inducible characteristics, these six mutations fall into four phenotypic classes: (i) an FT class, with either inhibition of all three analyzed functions at 42 degrees C (recA2283), preferable inhibition at 42 degrees C of recombination and the SOS response (recA2278), or inhibition at 42 degrees C of only recombination (recA2278-5); (ii) a moderately deficient class (recA2277); (iii) a nondeficient class (recA2283E); and (iv) a mutation with a null phenotype (recA2284) . The recA2223 mutation generates an L-223-->M substitution in beta-strand 6 in a central domain of the RecA structure . This FT mutation shows preferable inhibition of the SOS response at 42 degrees C . The recA2183 mutation produces a K-183-->M substitution in alpha-helix F of the same domain . The Lys-183 position in the Escherichia coli RecA protein was found among positions which are important for interfilament interaction (R . M . Story, I . T . Weber, and T . A . Steitz, Nature (London) 355:318-325, 1992).

J Bacteriol, 1996 Apr, 178(7), 1962 - 70
Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage; Linderoth NA et al.; Filamentous phage f1 encodes protein IV (pIV), a protein essential for phage morphogenesis that localizes to the outer membrane of Escherichia coli, where it is found as a multimer of 10 to 12 subunits . Introduction of internal His or Strep affinity tags at different sites in pIV interfered with its function to a variable extent . A spontaneous second-site suppressor mutation in gene IV allowed several different insertion mutants to function . The identical mutation was also isolated as a suppressor of a multimerization-defective missense mutation . A high-molecular-mass pIV species is the predominant form of pIV present in cells . This species is stable in 4% sodium dodecyl sulfate at temperatures up to 65 degrees C and is largely preserved at 100 degrees C in Laemmli protein sample buffer containing 4% sodium dodecyl sulfate . The suppressor mutation makes the high-molecular-mass form of wild-type pIV extremely resistant to dissociation, and it stabilizes the high-molecular-mass form of several mutant pIV proteins to extents that correlate with their level of function . Mixed multimers of pIV(f1) and pIV(Ike) also remain associated during heating in sodium dodecyl sulfate-containing buffers . Thus, sodium dodecyl sulfate- and heat-resistant high-molecular-mass pIV is derived from pIV multimer and reflects the physiologically relevant form of the protein essential for assembly-export.

J Bacteriol, 1996 Apr, 178(7), 1936 - 45
Identification, nucleotide sequence, and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon; Jovanovic G et al.; The phage shock protein (psp) operon (pspABCE) of Escherichia coli is strongly induced in response to a variety of stressful conditions or agents such as filamentous phage infection, ethanol treatment, osmotic shock, heat shock, and prolonged incubation in stationary phase . Transcription of the psp operon is driven from a sigma54 promoter and stimulated by integration host factor . We report here the identification of a transcriptional activator gene, designated pspF, which controls expression of the psp operon in E . coli . The pspF gene was identified by random miniTn10-tet transposon mutagenesis . Insertion of the transposon into the pspF gene abolished sigma54-dependent induction of the psp operon . The pspF gene is closely linked to the psp operon and is divergently transcribed from one major and two minor sigma 70 promoters, pspF encodes a 37-kDa protein which belongs to the enhancer-binding protein family of sigma54 transcriptional activators . PspF contains a catalytic domain, which in other sigma54 activators would be the central domain, and a C-terminal DNA-binding domain but entirely lacks an N-terminal regulatory domain and is constitutively active . The insertion mutant pspF::mTn10-tet (pspF877) encodes a truncated protein (PspF delta HTH) that lacks the DNA-binding helix-turn-helix (HTH) motif . Although the central catalytic domain is intact, PspF delta HTH at physiological concentration cannot activate psp expression . In the absence of inducing stimuli, multicopy-plasmid-borne PspF or PspF delta HTH overcomes repression of the psp operon mediated by the negative regulator PspA.

J Bacteriol, 1996 Apr, 178(7), 1919 - 27
Regulation of a heat shock sigma32 homolog in Caulobacter crescentus; Reisenauer A et al.; High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease . We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase sigma32 subunit, the sigma factor responsible for the transcription of heat shock promoters . The C . crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E . coli sigma32 and cross-reacts with a monoclonal antibody to E . coli sigma32 . Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E . coli rpoH deletion mutant with the C . crescentus rpoH gene . Immunoblot analysis showed a transient rise in sigma32 levels after a temperature shift from 30 to 42 degrees C similar to that described for E . coli . In addition, increasing the cellular content of sigma32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C . crescentus cultures grown at 30 degrees C . The C . crescentus rpoH gene was transcribed from either of two heat shock consensus promoters . rpoH transcription and sigma32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to sigma32 expression in this organism . Both the rpoH gene and sigma32 protein were expressed constitutively throughout the cell cycle at 30 degrees C . The isolation of rpoH provides an important tool for future studies of the role of sigma32 in the normal physiology of C . crescentus.

J Bacteriol, 1996 Apr, 178(7), 1903 - 7
A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains; Franco AV et al.; The modal distribution of O-antigen chain length is determined by the Wzz (Cld/Rol) protein in those cases in which it has been studied . The system of O-antigen synthesis in Escherichia coli serotypes O8 and O9 is different from that reported for most other bacteria, and chain length distribution is thought not to be determined by a Wzz protein . We report the existence in E . coli O8 and O9 strains of wzz genes which are very similar to and have sequences within the range of variation of those which determine the chain length of typical O antigens . We also find that wzz genes previously identified by their effect on O-antigen chain length, when cloned and transferred to O8 and O9 strains, affect the chain length of a capsule-related form of LPS, K(LPS) . We conclude that in at least some O8 and O9 strains there is a wzz gene which controls the chain length of K(LPS) but has no effect on the O8 or O9 antigen.

Infect Immun, 1996 Apr, 64(4), 1461 - 6
In vivo induction of apoptosis and immune responses in mice by administration of lipopolysaccharide from Porphyromonas gingivalis; Isogai E et al.; In vivo administration of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to mice induced apoptosis before a specific immune response . Apoptosis was associated with the expression of immunoglobulin and Ia on B cells and of CD5 and several markers on T cells . Apoptosis peaked in the spleen and lymph nodes on day 2, and the second peak occurred in the thymus on day 9 . Tumor necrosis factor alpha (TNF-alpha) could mediate apoptosis, because the serum TNF-alpha levels were significantly higher than those of controls at 1 day before apoptosis and recombinant murine TNF-alpha induced apoptosis . The apoptosis induced by Pg-LPS was similar to that induced by Escherichia coli LPS in its basic manner, but it was unique in the response of thymus T cells . It was suggested that Pg-LPS could induce apoptosis for the elimination of early nonspecific activated lymphocytes.

Infect Immun, 1996 Apr, 64(4), 1400 - 6
Mycobacterium tuberculosis invades and replicates within type II alveolar cells; Bermudez LE et al.; Although Mycobacterium tuberculosis is assumed to infect primarily alveolar macrophages after being aspirated into the lung in aerosol form, it is plausible to hypothesize that M . tuberculosis can come in contact with alveolar epithelial cells upon arrival into the alveolar space . Therefore, as a first step toward investigation of the interaction between M . tuberculosis and alveolar epithelial cells, we examined the ability of M . tuberculosis to bind to and invade alveolar epithelial cells in vitro . The H37Rv and H37Ra strains of M . tuberculosis were cultured to mid-log phase and used in both adherence and invasion assays . The A549 human type II alveolar cell line was cultured to confluence in RPMI 1640 supplemented with 5% fetal bovine serum, L-glutamine, and nonessential amino acids . H37Rv was more efficient in entering A549 cells than H37Ra, Mycobacterium avium, and Escherichia coli Hb101, and nonpiliated strain (4.7% +/- 1.0% of the initial inoculum in 2 h compared with 3.1% +/- 0.8%, 2.1% +/- 0.9%, and 0.03% +/- 0.0%, respectively) . The invasion was more efficient at 37 degrees C than 30 degrees C (4.7% +/- 1.0% compared with 2.3% +/- 0.8%) . H37Rv and H37Ra were both capable of multiplying intracellularly at a similar ration over 4 days . Binding was inhibited up to 55.7% by anti-CD51 antibody (antivitronectin receptor), up to 55% with anti-CD29 antibody (beta(1) integrin), and 79% with both antibodies used together . Update of M . tuberculosis H37Rv was microtubule and microfilament dependent . It was inhibited by 6l.4% in the presence of 10 micron colchicine and by 72.3% in the presence of 3 micron cytochalasin D, suggesting two separate pathways for uptake . Our results show that M . tuberculosis is capable of invading type II alveolar epithelial cells and raise the possibility that invasion of alveolar epithelial cells is associated with the pathogenesis of lung infection.

Infect Immun, 1996 Apr, 64(4), 1233 - 9
The structural gene encoding human enterotoxigenic Escherichia coli PCFO20 is homologous to that for porcine 987P; Viboud GI et al.; Putative colonization factor PCFO20 was recently identified in an enterotoxigenic Escherichia coli (ETEC) strain of serogroup O20 isolated from a child with diarrhea in Argentina . The gene encoding the structural subunit of PCFO20 fimbriae, fotA, was cloned from strain ARG-2 in the expression phage vector lambda ZAP Express . One positive clone, pGV29, that carried a 3.3-kb fragment was identified on the basis of fimbrillin production by using a monospecific rabbit anti-PCFO20 serum . Nucleotide sequencing of a 1.3-kb Sau3A-ClaI fragment of the subclone pGV292 containing the region coding for PCFO20 fimbrillin revealed two open reading frames of which one was complete . A western blot (immunoblot) showed that the cloned protein, FotA, migrated like the PCFO20 fimbrial subunit protein did . Fimbriae were not detected on the surface of E . coli host bacteria containing pGV292 or pGV29, suggesting that the genes needed for assembly of PCFO20 fimbriae are lacking in both clones . The fotA gene encodes a 20,574-Da prefimbrillin protein which contains a 21-amino-acid signal sequence; the mature protein has a size of 18.1 kDa . The subunit protein FotA was found to be more homologous to the subunit of porcine 987P than to any fimbrial subunit produced by human ETEC . Alignments of the amino acid sequences of the two proteins indicate that they are partly identical, with an overall similarity of 82% . FotA fimbrillin was shown to be transported and assembled by the fimbria assembly machinery in porcine ETEC strain 987 . PCFO20 and 987P may have evolved from a common ancestral gene . They are immunologically related but have affinity for different host cell receptors, since PCFO20-producing bacteria do not bind to neonatal piglet enterocytes.

Nat Struct Biol, 1996 Apr, 3(4), 355 - 63
Snapshot of an enzyme reaction intermediate in the structure of the ATP-Mg2+-oxalate ternary complex of Escherichia coli PEP carboxykinase; Tari LW et al.; We report the 1.8 A crystal structure of adenosine triphosphate (ATP)-magnesium-oxalate bound phosphoenolpyruvate carboxykinase (PCK) from Escherichia coli . ATP binding induces a 20 degree hinge-like rotation of the N- and C-terminal domains which closes the active-site cleft . PCK possesses a novel nucleotide-binding fold, particularly in the adenine-binding region, where the formation of a cis backbone torsion angle in a loop glycine residue promotes intimate contacts between the adenine-binding loop and adenine, while stabilizing a syn conformation of the base . This complex represents a reaction intermediate analogue along the pathway of the conversion of oxaloacetate to phosphoenolpyruvate, and provides insight into the mechanistic details of the chemical reaction catalysed by this enzyme.

Brain Res Dev Brain Res, 1996 Mar 29, 92(1), 1 - 9
Cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences in transgenic mouse embryos; Leconte L et al.; Peripherin is a neuron-specific type III intermediate filament protein expressed in well-defined populations of neurons projecting towards peripheral targets . To investigate the molecular mechanisms by which a gene is expressed in a specific subset of neurons, we used a transgenic approach in order to define peripherin gene sequences that are necessary for cell-type specific expression . Transgenic mice carrying different various genomic regions of the mouse peripherin gene fused to the Escherichia coli lacZ reporter gene were generated . We used three different peripherin/lacZ constructs containing either 5.8 kb upstream sequences, or both 5.8 kb upstream and 1.1 kb intragenic sequences, or 1.1 kb intragenic sequences associated with an heterologous promoter . Analysis of lacZ gene expression in transgenic mouse embryos showed that cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences . Analysis of transgenic mouse lines expressing the construct containing both upstream and intragenic sequences showed that this transgene contains all regulatory elements essential for both spatial and temporal expression of the mouse peripherin gene during embryogenesis . Furthermore, lacZ+ positive cells isolated from these transgenic lines by fluorescence-activated cell sorting (FACS) can be stained with a peripherin antibody, demonstrating that the transgene containing both upstream and intragenic sequences is expressed in peripherin neurons . These mouse peripherin upstream and intragenic sequences can now be used to identify cis-acting regulatory elements and transcription factors involved in peripherin gene regulation.

J Biol Chem, 1996 Mar 29, 271(13), 7851 - 9
Overproduction of DNA cytosine methyltransferases causes methylation and C --> T mutations at non-canonical sites; Bandaru B et al.; Multicopy clones of Escherichia coli cytosine methyltransferases Dcm and EcoRII methylase (M . EcoRII) cause an approximately 50-fold increase in C --> T mutations at their canonical site of methylation, 5'-CmeCAGG (meC is 5-methylcytosine) . These plasmids also cause transition mutations at the second cytosine in the sequences CCGGG at approximately 10-fold lower frequency . Similarly, M . HpaII was found to cause a significant increase in C --> T mutations at a CCAG site, in addition to causing mutations at its canonical site of methylation, CCGG . Using a plasmid that substantially overproduces M . EcoRII, in vivo methylation at CCSGG (S is C or G) and other non-canonical sites could be detected using a gel electrophoretic assay . There is a direct correlation between the level of M . EcoRII activity in cells, the extent of methylation at non-canonical sites and frequency of mutations at these same sites . Overproduction of M . EcoRII in cells also causes degradation of DNA and induction of the SOS response . In vitro, M . EcoRII methylates an oligonucleotide duplex containing a CCGGG site at a slow rate, suggesting that overproduction of the enzyme is essential for significant amounts of such methylation to occur . Together these results show that cytosine methyltransferases occasionally methylate cellular DNA at non-canonical sites and suggest that in E . coli, methylation-specific restriction systems and sequence specificity of the DNA mismatch correction systems may have evolved to accommodate this fact . These results also suggest that mutational effects of cytosine methyltransferases may be much broader than previously imagined.

J Biol Chem, 1996 Mar 29, 271(13), 7788 - 95
The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency; Keyt BA et al.; Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells . VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia . VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing . VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity . The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF . We characterized the proteolysis of VEGF by plasmin and other proteases . Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments . The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis . Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin . Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111 . A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165 . The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors . The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM) . In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively . Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165 . The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.

J Biol Chem, 1996 Mar 29, 271(13), 7752 - 7
Identification of a consensus cyclin-dependent kinase phosphorylation site unique to the nuclear form of human deoxyuridine triphosphate nucleotidohydrolase; Ladner RD et al.; In the preceding report (Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J . (1996) J . Biol . Chem . 271, 7745-7751), we identified two distinct isoforms of dUTPase in human cells . These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M) . The proteins are nearly identical, differing only in a short region of their amino termini . Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article) . In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S . (1990) J . Cell . Biochem . 43, 339-353) . To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken . {32P}Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated . Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein) . Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo . Analysis of the wild type and Ser-11 --> Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro . Additionally, experiments with the Ser-11 --> Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase . The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34(cdc2) . Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34(cdc2) . Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo.

J Biol Chem, 1996 Mar 29, 271(13), 7635 - 9
A family of genes coding for two serologically distinct chicken interferons; Sick C et al.; Southern blot analysis and screening of a genomic lambda phage library with the previously cloned chicken interferon (IFN) cDNA indicated that the chicken genome contains at least 10 IFN genes . A particularly strongly hybridizing phage clone that we analyzed in more detail carried a head to tail arrangement of three intron-less IFN genes that differed from each other and from the cloned chicken IFN cDNA by only a few base changes . The primary translation products of these three IFN genes consist of 193 amino acids, and the mature proteins are composed of 162 amino acids . All three genes of this IFN family, designated IFN1, yielded active chicken IFN when expressed individually in transfected COS7 cells . A weakly hybridizing phage clone contained an additional intron-less chicken IFN gene, designated IFN2, whose product was 57% identical to chicken IFN1 . Southern blot analysis suggested that the chicken genome contains a single IFN2 gene . The primary translation product of IFN2 consists of 203 amino acids, and the mature protein is composed of 176 amino acids . Purified recombinant chicken IFN2 from Escherichia coli had a specific antiviral activity of about 10(6) units/mg, which was about 20-fold lower than that of chicken IFN1 purified in parallel . The antiviral activity of chicken IFN2 from E . coli or from transfected COS7 cells could not be neutralized by antiserum to recombinant chicken IFN1 . Thus, like mammals, the chicken has a large number of type I IFN genes that code for at least two serologically distinct antiviral activities.

J Biol Chem, 1996 Mar 29, 271(13), 7630 - 4
Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library; de Kruif J et al.; This report describes the construction of leucine zipper-based dimerization cassettes for the conversion of recombinant monomeric scFv antibody fragments to bivalent and bispecific dimers . A truncated murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned into four scFv fragments previously isolated from a synthetic antibody phage display library . Cysteine residues flanking the zipper region were introduced to covalently link dimerized scFv fragments . The secreted fusion proteins were shown to spontaneously and efficiently form stable Fos-Fos or Jun-Jun homodimers in the Escherichia coli periplasm at levels comparable to their monovalent counterparts . The bivalent (scFv)2 fragments performed well in enzyme-linked immunosorbent assay, flowcytometric, and immunohistochemical analysis . Fos and Jun homodimer (scFv)2 antibodies with different specificities could be reduced, reshuffled, and reoxidized to form preparations of functional bispecific (scFv)2 Fos-Jun heterodimers . These Fos and Jun fusion protein cassettes provide a universal basis for the construction of dimeric scFv antibodies with enhanced avidity or dual specificity.

J Biol Chem, 1996 Mar 29, 271(13), 7568 - 73
Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers; Hamman BD et al.; The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12 . Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe . The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants . Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99) . The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains . The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12 . It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers . Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band . This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.

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