Escherichia coli

FEMS Microbiol Lett, 1997 Nov 1, 156(1), 69 - 78
Identification and cloning of the Mycobacterium avium folA gene, required for dihydrofolate reductase activity; Zywno-van Ginkel S et al.; Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state . In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time . It has an open reading frame of 543 bp with a G + C content of 73% . The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases . Recombinant M . avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence:

FEMS Microbiol Lett, 1997 Nov 1, 156(1), 49 - 53
Insertion site of the locus of enterocyte effacement in enteropathogenic and enterohemorrhagic Escherichia coli differs in relation to the clonal phylogeny of the strains; Wieler LH et al.; The locus of enterocyte effacement pathogenicity island confers the attaching and effacing histopathology on epithelial cells infected with enteropathogenic and enterohemorrhagic Escherichia coli . We investigated the site of insertion of the locus of enterocyte effacement in E . coli strains in relation to their evolution based on conservation of housekeeping proteins in these strains . The results indicate that the insertion site of the locus of enterocyte effacement varies according to the evolutionary lineage, suggesting that it has inserted at multiple times and sites during the evolution of these pathogens.

FEMS Microbiol Lett, 1997 Nov 1, 156(1), 21 - 9
Protein secretion in Streptomyces griseus N2-3-11: characterization of the secA gene and its growth phase-dependent expression; Pohling S et al.; The chromosomal region encoding the secA gene of Streptomyces griseus N2-3-11 was cloned and analyzed . The secA gene encodes a polypeptide of 939 aa with a molecular mass of 105 kDa . The growth defect of temperature sensitive Escherichia coli secA mutants was not restored by the S . griseus SecA . The secA promoter was analyzed and the transcriptional start point of the gene was determined . Northern blot and Western blot analyses revealed a growth phase dependent secA expression . The integration of an additional copy of the S . griseus secA gene into the genome of S . lividans TK23 had no visible effect on the efficiency of protein secretion.

Stem Cells, 1997, 15 Suppl 1, 113 - 9; discussion 120
Frequency of point mutations in the gene for the G-CSF receptor in patients with chronic neutropenia undergoing G-CSF therapy; Tidow N et al.; Point mutations in the gene for the G-CSF receptor have been reported previously in a subgroup of patients with severe congenital neutropenia . Here, we investigated the frequency of these specific G-CSF receptor mutations in patients with neutropenic disorders undergoing treatment with recombinant human (r-metHu)G-CSF (Filgrastim) . Nucleotides 2306 to 2561, including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene, were amplified by reverse transcriptase-polymerase chain reaction, and DNA was sequenced directly and after transformation in E . coli . Four of 30 patients with severe congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene . Two of the four patients with a mutated G-CSF receptor developed acute myeloid leukemia secondary to congenital neutropenia . G-CSF receptor analyses were performed in myeloid cells taken at different time points in the four patients with the mutated receptor, and no correlation between occurrence of the mutation and time or dose of r-metHuG-CSF treatment was found . No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 26 congenital neutropenia patients . Additionally, no G-CSF receptor point mutations could be seen in neutrophils, blood and bone marrow mononuclear cells from patients with cyclic or idiopathic neutropenia, and bone marrow mononuclear cells from patients suffering from severe aplastic anemia . Similar results were obtained by Touw et al., demonstrating that five out of 25 patients with congenital neutropenia reveal G-CSF receptor mutations . These data show that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur only in a subgroup of severe congenital neutropenia patients . Furthermore, our data suggest that the described G-CSF receptor point mutations are not correlated with the start, duration or doses of r-metHuG-CSF treatment, but might result from genetic instability in the G-CSF receptor gene in severe congenital neutropenia.

J Inflamm, 1998, 48(1), 1 - 12
Neutrophil structural changes associated with chronic endotoxemia and lung injury; Klut ME et al.; Previous studies showed that chronic endotoxemia induces thickening of the alveolar wall of rabbits . The present study examines cellular changes associated with this process and attempts to define the role of PMN in this response . Rabbits received i.v . injections of either Escherichia coli lipopolysaccharide (LPS) or saline (control), 2-3 times weekly, for 28 weeks . Peripheral blood mature and immature polymorphonuclear leukocyte (PMN) cell counts were determined on Wright-stained blood smears . Lung histological analysis was performed by both light and electron microscopy . FITC-Maclura pomifera was used to identify type II cells and diaminobenzidine tetrahydrochloride-H2O2 was employed to localize peroxidase . The results show that the LPS-induced neutrophilia is associated with an increase in the circulating band cells which is consistent with active bone marrow release . PMN in the pulmonary microvessels display structural features characteristic of phagocytosis and active macromolecule synthesis . Endothelial cells, adjacent to these PMN, show numerous coated pits and large inclusions suggestive of endocytosis . The LPS-induced thickening of the alveolar wall is associated with leukocyte migration into the interstitial and alveolar spaces . Some interstitial PMN are fragmented and surrounded by dispersed elements of the connective tissue, while others appear activated and are closely associated with hyperactive fibroblasts and alveolar type II cells . The number of alveolar type II cells has increased twofold . These results show that chronic endotoxemia in rabbits causes structural changes in PMN, endothelium, interstitium, and epithelium . PMN structural changes are consistent with enhanced functional properties and their close association with modified regions of the lung parenchyma suggest that PMN play an important role in the process of this lung injury and repair.

J Mol Biol, 1997 Oct 17, 273(1), 299 - 316
Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface; Nohaile M et al.; The bacterial enhancer-binding protein NtrC activates transcription when phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substitutions . The N-terminal domain of NtrC, which acts positively on the remainder of the protein, is homologous to a large family of signal transduction domains called receiver domains . Phosphorylation of an aspartate residue in a receiver domain modulates the function of a downstream target, but the accompanying structural changes are not clear . In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying constitutively activating substitutions . Combinations of such substitutions resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein . Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site . Interestingly, the activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face . Thus, the active site and the 3445 face appear to interact . Implications with respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainder of NtrC are discussed.

J Mol Biol, 1997 Oct 17, 273(1), 256 - 68
Structure and mechanism of L-fucose isomerase from Escherichia coli; Seemann JE et al.; The three-dimensional structure of L-fucose isomerase from Escherichia coli has been determined by X-ray crystallography at 2.5 A resolution . This ketol isomerase converts the aldose L-fucose into the corresponding ketose L-fuculose using Mn2+ as a cofactor . Being a hexamer with 64,976 Da per subunit, L-fucose isomerase is the largest structurally known ketol isomerase . The enzyme shows neither sequence nor structural similarity with other ketol isomerases . The hexamer obeys D3 symmetry and forms the crystallographic asymmetric unit . The strict and favorably oriented local symmetry allowed for a computational phase extension from 7.3 A to 2.5 A resolution . The structure was solved with an L-fucitol molecule bound to the catalytic center such that the hydroxyl groups at positions 1 and 2 are ligands of the manganese ion . Most likely, L-fucitol mimics a bound L-fucose molecule in its open chain form . The protein environment suggests strongly that the reaction belongs to the ene-diol type.

J Mol Biol, 1997 Oct 17, 273(1), 226 - 37
The 1.6 A crystal structure of the AraC sugar-binding and dimerization domain complexed with D-fucose; Soisson SM et al.; The crystal structure of the sugar-binding and dimerization domain of the Escherichia coli gene regulatory protein, AraC, has been determined in complex with the competitive inhibitor D-fucose at pH 5.5 to a resolution of 1.6 A . An in-depth analysis shows that the structural basis for AraC carbohydrate specificity arises from the precise arrangement of hydrogen bond-forming protein side-chains around the bound sugar molecule . van der Waals interactions also contribute to the epimeric and anomeric selectivity of the protein . The methyl group of D-fucose is accommodated by small side-chain movements in the sugar-binding site that result in a slight distortion in the positioning of the amino-terminal arm . A comparison of this structure with the 1.5 A structure of AraC complexed with L-arabinose at neutral pH surprisingly revealed very small structural changes between the two complexes . Based on solution data, we suspect that the low pH used to crystallize the fucose complex affected the structure, and speculate about the nature of the changes between pH 5.5 and neutral pH and their implications for gene regulation by AraC . A comparison with the structurally unrelated E . coli periplasmic sugar-binding proteins reveals that conserved features of carbohydrate recognition are present, despite a complete lack of structural similarity between the two classes of proteins, suggesting convergent evolution of carbohydrate binding.

J Mol Biol, 1997 Oct 17, 273(1), 207 - 25
Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis; Perona JJ et al.; The structures of wild-type and mutant forms of the unliganded EcoRV endonuclease dimer have been determined at 2.4 A resolution in a new crystal lattice . Comparison of these structures with that of the free enzyme determined with different packing constraints shows that the conformations of the domain interfaces are not conserved between crystal forms . The unliganded enzyme and the enzyme-DNA complex delineate two distinct quaternary states separated by a 25 degrees intersubunit rotation, but considerable conformational heterogeneity, of the order of 10 degrees domain rotations, exists within each of these states . Comparison of the free enzyme structure between the two crystal forms further reveals that the C-terminal 28 amino acid residues are disordered and undergo an extensive local folding transition upon DNA binding . Introduction of the mutation T93A at the DNA-binding cleft causes large-scale effects on the protein conformation . Structural changes in the mutated unliganded enzyme propagate some 20 to 25 A to the dimerization interface and lead to a rearrangement of monomer subunits . Comparative analysis of these structures, a new structure of the enzyme cocrystallized with DNA and calcium ions, and previously determined cocrystal structures suggests important roles for a number of amino acid residues in facilitating the intersubunit motions and local folding transitions . In particular, the T93A structure reveals a pathway through the protein, by which DNA-binding may cause the domain movements required for proper alignment of catalytic groups . The key active-site residue Glu45 is located on a flexible helix inside this pathway, and this provides a direct means by which essential catalytic functions are coupled to the protein conformational change . It appears that indirect perturbation of the Glu45 conformation via an altered quaternary structure may be a contributing factor to the decreased catalytic efficiency of T93A, and this mechanism may also explain the diminished activities of other active site variants of EcoRV.

J Mol Biol, 1997 Oct 17, 273(1), 105 - 13
(GT)n repetitive tracts affect several stages of RecA-promoted recombination; Dutreix M; The effect of GT/CA dinucleotide repeat tracts on RecA-dependent homologous recombination was examined in vitro . By measuring the binding of RecA protein to oligonucleotides containing GT or CA repeats using the surface plasmon resonance (BIAcore), we show that the efficiency of RecA protein binding is sequence dependent: the protein binds to non-repetitive, poly(CA) or poly(GT) sequences with an increasing affinity . This preferential binding to repetitive sequences is specific for RecA protein and is not observed with the single-strand DNA binding (SSB) protein . Despite the fact that RecA filaments formed on GT tracts efficiently bind duplex DNAs, they are unable to promote stable joint formation . Moreover, strand exchange between a duplex DNA and a fully homologous single-stranded DNA (ssDNA) containing dinucleotide repeats, is inhibited as a function of the length of the repetitive tract . The number of molecules which underwent a complete strand exchange decreased from 100% to 80% and 30% for DNA containing seven, 16 and 39 GT repeats, respectively . The inhibition is less pronounced when the ssDNA contains CA instead of GT dinucleotide repeats . We propose that the high affinity of RecA protein for (CA)n or (GT)n tracts prevents strand exchange from progressing across such sequences . Thus, our results suggest that repetitive tracts of dinucleotides CA/GT could influence recombinational activity potentially leading to an increase in genomic rearrangements.

J Mol Biol, 1997 Oct 17, 273(1), 93 - 104
Variation in HU composition during growth of Escherichia coli: the heterodimer is required for long term survival; Claret L et al.; The histone-like dimeric HU protein of Escherichia coli is encoded by two closely related genes, hupA and hupB . We show here that expression from the single hupA promoter and from the three hupB promoters varies during growth phase . The weak hupB-P4 promoter is active immediately after dilution . Transcription of the hupA gene is activated early in logarithmic phase . A little later, at mid to late exponential phase, RNA originating at the hupB-P2 promoter is detected . The hupB-P3 promoter is activated last when the cells enter stationary phase . Although the hup mRNAs are unstable, the HU protein is very stable so that the variations in the mRNAs synthesis are reflected in the level of the two HU subunits and in the composition of HU dimers . Cells growing exponentially contain a mixture of homodimeric alpha 2 and heterodimeric alpha beta but no beta 2 is detected . In stationary cells, the predominant form is the heterodimer alpha beta . The presence of the heterodimeric form is required for optimal survival of E . coli after prolonged starvation . The three forms of HU are not equivalent, since beta 2 is incapable of promoting formation of DNA supercoiling like alpha beta and alpha 2 do . The putative roles of each form of HU are discussed.

J Mol Biol, 1997 Oct 17, 273(1), 75 - 83
Role of DNA supercoiling and rpoS sigma factor in the osmotic and growth phase-dependent induction of the gene osmE of Escherichia coli K12; Conter A et al.; Transcription of the gene osmE of Escherichia coli is osmotically inducible and regulated by the growth phase . In a medium of low osmotic pressure, expression of osmE is induced at the onset of stationary phase . At elevated osmotic pressure, a biphasic induction pattern is observed . The first step occurs during exponential phase, and this is followed by a strong induction at the onset of stationary phase . Both steps appear to result from stimulation of transcription at the same promoter, osmEp . In the absence of sigma s, the stationary phase sigma factor encoded by rpoS, osmEp stationary phase induction is abolished, while the osmotic effect is still observed . Mutations that compensate for the absence of sigma s mapped to the gene topA . The effect of such mutation and of novobiocin, an inhibitor of DNA gyrase, suggest that changes in DNA supercoiling are involved in the osmotic induction of osmEp . In addition, modulation of the supercoiling level of a reporter plasmid was observed during growth in rich media . The kinetics of osmEp transcription are discussed in light of the variations of DNA supercoiling.

J Mol Biol, 1997 Oct 17, 273(1), 61 - 74
The GCN4 leucine zipper can functionally substitute for the heat shock transcription factor's trimerization domain; Drees BL et al.; The heat shock transcription factor (HSF) is the only known sequence-specific, homotrimeric DNA-binding protein . HSF binds to a DNA recognition site called a heat shock element (HSE), which contains varying numbers of nGAAn units ("GAA boxes") arranged in inverted repeats . To investigate the role of trimerization on HSF's DNA-binding properties, we replaced the trimerization domain, which self-assembles to form a three-stranded alpha-helical coiled coil, with the GCN4 leucine zipper, which forms a two-stranded alpha-helical coiled coil . Surprisingly, this substitution did not effect the ability of HSF to function in vivo . Biochemical studies of an HSF-leucine zipper chimera in comparison to an HSF truncation show that the HSF-leucine zipper chimera, though dimeric in solution and dimeric when bound to a two-box HSE, forms a trimeric complex when bound to a three-box HSE . The ability to form trimers depends on the presence of three contiguous GAA boxes present in inverted repeats . The proximity of the leucine zippers due to the orientation of the binding sites suggests that the leucine zippers might be forming a three-stranded coiled coil and several experiments lend support to this model . The ability of the leucine zipper to change oligomeric states in context might explain why the leucine zipper can replace the trimerization domain of HSF in vivo.

J Mol Biol, 1997 Oct 17, 273(1), 38 - 51
Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding; Franch T et al.; The hok/sok locus of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells . The locus specifies two RNAs, hok mRNA and Sok antisense RNA . The post-segregational killing mediated by hok/sok is governed by a complicated control mechanism that involves both post-transcriptional inhibition of translation by Sok-RNA and activation of hok translation by mRNA 3' processing . Sok-RNA inhibits translation of a reading frame (mok) that overlaps with hok, and translation of hok is coupled to translation of mok . In the inactive full-length hok mRNA, the translational activator element at the mRNA 5'-end (tac) is sequestered by the fold-back-inhibitory element located at the mRNA 3'-end (fbi) . The 5' to 3' pairing locks the RNA in an inert configuration in which the SDmok and Sok-RNA target regions are sequestered . Here we show that the 3' processing leads to major structural rearrangements in the mRNA 5'-end . The structure of the refolded RNA explains activation of translation and antisense RNA binding . The refolded RNA contains an antisense RNA target stem-loop that presents the target nucleotides in a single-stranded conformation . The stem of the target hairpin contains SDmok and AUGmok in a paired configuration . Using toeprinting analysis, we show that this pairing keeps SDmok in an accessible configuration . Furthermore, a mutational analysis shows that an internal loop in the target stem is prerequisite for efficient translation and antisense RNA binding.

J Mol Biol, 1997 Oct 17, 273(1), 26 - 37
Programmed cell death by hok/sok of plasmid R1: coupled nucleotide covariations reveal a phylogenetically conserved folding pathway in the hok family of mRNAs; Gultyaev AP et al.; The hok/sok system of plasmid R1 mediates plasmid maintenance by killing of plasmid-free cells . Translation of the stable toxin-encoding hok mRNA is repressed by the unstable Sok antisense RNA . Using genetic algorithm simulations and phylogenetic comparisons, we analyse five plasmid-encoded and two chromosome-encoded hok-homologous mRNAs . A similar folding pathway was found for all mRNAs . Metastable hairpins at the very 5'-ends of the mRNAs were predicted to prevent the formation of structures required for translation and antisense RNA binding . Thus the folding of the mRNA 5'-ends appears to explain the apparent inactivity of the nascent transcripts . In the full-length mRNAs, long-range 5' to 3' interactions were predicted in all cases . The 5' to 3' interactions lock the mRNAs in inactive configurations . Translation of the mRNAs is activated by 3' exonucleolytic processing . Simulation of the 3' processing predicted that it triggers rearrangements of the mRNA 5'-ends with the formation of translational activator and antisense RNA target hairpins . Alignment of the mRNA sequences revealed a large number of nucleotide covariations that support the existence of the proposed secondary structures . Furthermore, coupled covariations support the folding pathway and provide evidence that the mRNA 5'-ends pair with three different partners during the proposed series of dynamic RNA rearrangements.

J Mol Biol, 1997 Oct 17, 273(1), 19 - 25
Solution structure of the I gamma subdomain of the Mu end DNA-binding domain of phage Mu transposase; Clubb RT et al.; The MuA transposase of phase Mu is a large modular protein that plays a central role in transposition . We show that the Mu end DNA-binding domain, I beta gamma, which is responsible for binding the DNA attachment sites at each end of the Mu genome, comprises two subdomains, I beta and I gamma, that are structurally autonomous and do not interact with each other in the absence of DNA . The solution structure of the I gamma subdomain has been determined by multidimensional NMR spectroscopy . The structure of I gamma comprises a four helix bundle and, despite the absence of any significant sequence identity, the topology of the first three helices is very similar to that of the homeodomain family of helix-turn-helix DNA-binding proteins . The helix-turn-helix motif of I gamma, however, differs from that of the homeodomains in so far as the loop is longer and the second helix is shorter, reminiscent of that in the POU-specific domain.

Development, 1997 Oct, 124(19), 3747 - 54
Integration of the head and trunk segmentation systems controls cephalic furrow formation in Drosophila; Vincent A et al.; Genetic and molecular analyses of patterning of the Drosophila embryo have shown that the process of segmentation of the head is fundamentally different from the process of segmentation of the trunk . The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems . We report here that the initial step in CF formation is a change in shape and apical positioning of a single row of cells . The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped (eve) . Re-examination of the btd and eve phenotypes in live embryos indicated that both genes are required for CF formation . Further, Eve expression in initiator cells was found to be dependent upon btd activity . The control of eve expression by btd in these cells is the first indication of a new level of integrated regulation that interfaces the head and trunk segmentation systems . In conjunction with previous data on the btd and eve embryonic phenotypes, our results suggest that interaction between these two genes both controls initiation of a specific morphogenetic movement that separates two morphogenetic fields and contributes to patterning the hinge region that demarcates the procephalon from the segmented germ band.

J Gen Virol, 1997 Nov, 78 ( Pt 11), 2923 - 31
The product of the US10 gene of herpes simplex virus type 1 is a capsid/tegument-associated phosphoprotein which copurifies with the nuclear matrix; Yamada H et al.; We have identified the herpes simplex virus type 1 (HSV-1) US10 gene product using rabbit polyclonal antisera raised against a recombinant 6xHis-US10 fusion protein expressed in Escherichia coli . The antiserum reacted specifically with 34 and 36 kDa proteins in HSV-1 KOS-infected cells as shown by Western blotting and immunoprecipitation experiments . The 36 kDa protein was immunoprecipitated with the US10 antiserum from 32P-labelled lysates of Vero cells infected with HSV-1 KOS, demonstrating that the US10 protein was phosphorylated . Indirect immunofluorescence studies localized the US10 protein mainly to nuclei as large discrete particles at later times post-infection (p.i.), and nuclear fractionation studies revealed that the protein was tightly associated with the nuclear matrix . Moreover, analysis of isolated intracellular capsids showed that both phosphorylated and unphosphorylated forms of the US10 product were also associated with the capsid/tegument . These results indicate that the US10 gene of HSV-1 encodes a capsid/tegument-associated phosphoprotein which copurifies with the nuclear matrix.

FASEB J, 1997 Nov, 11(13), 1169 - 76
The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin mediated; Fabbrini MS et al.; In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized . To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3) . The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity . Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR . Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP . We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways . Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors.

FASEB J, 1997 Nov, 11(13), 1153 - 6
Use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve mRNA and its protein product in one gel; Gao W et al.; We demonstrate that it is possible to simultaneously resolve both an mRNA and its protein product by electrophoresis in a single SDS-polyacrylamide gel by using double labeling with {32P}H3PO4 and {35S}methionine, and an elongated 5% stacking gel atop the 10% resolving gel . The mRNA is resolved in the 5% gel; the protein, as expected, resolves in the 10% gel . Using a T7 expression system, we show that putative mRNA bands in the 5% gel are: 1) labeled only with 32P and not with 35S; 2) inducible with isopropylthiogalactopyranoside (needed to induce a T7 RNA polymerase gene under control of a lac promoter); 3) synthesized in the presence of rifampicin (T7 RNA polymerase is not inhibited by rifampicin); 4) degraded by base or RNase treatment; and 5) are largely resistant to DNase treatment . The mRNA bands were also evident in samples not treated with rifampicin . We used this technique to confirm previously published results that inhibition of expression by consecutive low-usage AGG arginine codons inserted near the 5' end of a test message in Escherichia coli is at the level of translation.

Crit Care Med, 1997 Nov, 25(11), 1874 - 80
Cardiovascular toxicity of human cross-linked hemoglobin in a rabbit endotoxemia model; Krishnamurti C et al.; OBJECTIVE: To determine the possible adverse effects of human cross-linked hemoglobin in endotoxemia . DESIGN: Prospective, controlled, laboratory trial . SETTING: Animal research laboratory . SUBJECTS: New Zealand white rabbits . INTERVENTIONS: Conscious rabbits received intravenous infusions of either lipopolysaccharide (LPS) alone (10 micrograms/kg, Escherichia coli 0111:B4), human hemoglobin cross-linked between the alpha chains (alpha alpha Hb, 0.7 g/kg), or both LPS and alpha alpha Hb . The cardiovascular effects of alpha alpha Hb and LPS as single agents or administered together were then studied in anesthetized rabbits . MEASUREMENTS AND MAIN RESULTS: Mortality in conscious animals that received alpha alpha Hb followed by LPS 4 hrs later (n = 5), or LPS and alpha alpha Hb at the same time (n = 6) was 60% and 67%, respectively . In anesthetized animals, infusion of both LPS and alpha alpha Hb (n = 6) resulted in hypoxia, lactic acidosis, ventricular arrhythmias, and decreased myocardial contractility and left ventricular pressure . In contrast, anesthetized rabbits that received alpha alpha Hb (n = 5) or LPS (n = 5) alone did not develop hypoxia, acidosis, alteration in myocardial contractility, or arrhythmias . Furthermore, death did not occur in any of the conscious animals that received either LPS (n = 7) or alpha alpha Hb (n = 4) as single agents . CONCLUSIONS: In an animal model of nonlethal endotoxemia, infusion of alpha alpha Hb significantly increases mortality . Our data suggest that mortality may be due to the acute increased cardiopulmonary toxicity of alpha alpha Hb in animals with underlying endotoxemia.

Arch Surg, 1997 Nov, 132(11), 1165 - 9; discussion 1170
Glutathione depletion prevents lipopolysaccharide-induced local skin inflammation; Jones JJ et al.; BACKGROUND: We have previously shown that the thiol-oxidizing agent diethyl maleate prevents lipopolysaccharide (LPS)-induced up-regulation of endothelial cell intercellular adhesion molecule-1 (ICAM-1) in vitro . OBJECTIVE: To determine the effect of glutathione depletion on the development of local skin inflammation in vivo, a model known to be dependent on ICAM-1 . DESIGN: Swiss Webster mice were injected with intradermal LPS (30 micrograms) or isotonic saline solution . INTERVENTION: Mice were pretreated for 1 hour with intraperitoneal diethyl maleate (6 mmol/kg) or corn oil vehicle . MAIN OUTCOME MEASURES: Injection sites were harvested after 12 and 24 hours and evaluated for changes in vascular permeability and histological characteristics . To determine the mechanism underlying our findings, we evaluated skin ICAM-1 immunohistochemistry, levels of ICAM-1 protein and messenger RNA (mRNA), and neutrophil CD11b expression at the 24-hour point . RESULTS: Diethyl maleate significantly decreased the skin permeability index in a dose-dependent fashion at 24 hours but not at 12 hours . Skin histological examination under light microscopy showed a marked LPS-induced neutrophil infiltration at 24 hours, which was inhibited with diethyl maleate pretreatment . Immunohistochemical examination showed that diethyl maleate reduced ICAM-1 expression . In keeping with the hypothesized mechanism, diethyl maleate attenuated the LPS-induced up-regulation of ICAM-1 mRNA by 44% . Diethyl maleate also slightly but insignificantly reduced CD11b expression in vivo . CONCLUSIONS: Diethyl maleate markedly attenuates LPS-induced dermal inflammation, primarily through a reduction in ICAM-1 protein and mRNA expression . These data suggest that manipulation of the intracellular redox state may have a beneficial role in neutrophil-mediated inflammation.

Emerg Infect Dis, 1997 Oct-Dec, 3(4), 483 - 7
Quantitative risk assessment: an emerging tool for emerging foodborne pathogens; Lammerding AM et al.; New challenges to the safety of the food supply require new strategies for evaluating and managing food safety risks . Changes in pathogens, food preparation, distribution, and consumption, and population immunity have the potential to adversely affect human health . Risk assessment offers a framework for predicting the impact of changes and trends on the provision of safe food . Risk assessment models facilitate the evaluation of active or passive changes in how foods are produced, processed, distributed, and consumed.

Arch Insect Biochem Physiol, 1997, 36(4), 251 - 71
Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae; Cui L et al.; The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts . Four related cysteine-rich polydnavirus gene have been identified in parasitized H . virescens larvae and grouped into a family . In this study, we investigated the expression and hemocyte targeting of the cysteine-rich VHv1.4 protein . Full-length and truncated VHv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera . In immunoblots the VHv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism . The VHv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted . The VHv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus . Digestion with endoglycosidases suggests that the VHv1.4 protein is glycosylated at multiple N-glycosylation sites . Immunofluorescence assays showed that the VHv1.4 protein binds to the hemocytes, most notably the granulocytes, in H . virescens larvae . After binding, the VHv1.4 protein was internalized, probably by endocytosis . Specific binding of the VHv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response.

Genes Cells, 1997 Jul, 2(7), 433 - 41
Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate; Kusano S et al.; BACKGROUND: RNA polymerase from the stationary growth phase cells of Escherichia coli is tightly associated with an acidic compound(s) and exhibits altered promoter selectivity, with reduced transcriptional activity of the genes highly expressed in the exponentially growing cells . Here we have examined the nature of the RNA polymerase-associated acidic compound(s) . RESULTS: RNA polymerase isolated from stationary-phase cells of E . coli was found to be tightly associated with inorganic polyphosphates (poly P), and cannot be dissociated even after chromatography on phosphocellulose or DNA-cellulose columns . Since RNA polymerase-poly P complexes reconstituted in vitro showed similar properties, poly P was not binding covalently . The inhibitory effects of poly P on transcription were examined using two forms of the holoenzyme, one containing sigma70, the major sigma for transcription of the genes expressed during exponential cell growth and the other containing sigma38, the principal sigma in the stationary growth phase . At low salt concentrations, poly P inhibited transcription by both Esigma70 and Esigma38 holoenzymes . With an increase in the concentration of potassium glutamate, the poly P inhibition was relieved . At high salt concentrations, the Esigma70 holoenzyme is not able to function, but the Esigma38 holoenzyme is however activated . CONCLUSIONS: These results show that poly P may play a role in the promoter selectivity control of RNA polymerase in E . coli which is growing under conditions of high osmolarity and in the stationary growth phase.

Biochim Biophys Acta, 1997 Sep 26, 1342(1), 28 - 36
Partial reconstitution of mammalian phosphoribosylpyrophosphate synthetase in Escherichia coli cells . Coexpression of catalytic subunits with the 39-kDa associated protein leads to formation of soluble multimeric complexes of various compositions; Ishijima S et al.; Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of 34-kDa catalytic subunits (PRS I and PRS II) and homologous 39- and 41-kDa proteins termed PRPP synthetase-associated proteins (PAPs) . While a negative regulatory role was indicated for PAPs, the physiological function of PAPs is less well understood . We attempted to prepare recombinant 39-kDa PAP (PAP39) and to reconstitute the enzyme complex . Free PAP39 was poorly expressed in Escherichia coli, while expression of protein fused with glutathione S-transferase was successful . The purified fusion protein had no PRPP synthetase activity, and bound to dissociated PRS I and PRS II, with a similar affinity . A free form of PAP39 prepared from the fusion protein formed insoluble aggregates . The enzyme complex was then partially reconstituted in situ by coexpression of PAP39 with PRS I or PRS II in E . coli cells . This coexpression led to formation of soluble complexes of various compositions, depending on the conditions . When the relative amount of PAP39 was higher, specific catalytic activities, in terms of the amount of the catalytic subunit, were lowered . PAP39 complexed with PRS I was more readily degraded by proteolysis than seen with PRS II, in vivo and in vitro . These results provide additional, strong evidence for that PAP39 has no catalytic activity in the enzyme complex, but does exert inhibitory effects in an amount-dependent manner, and that composition of the enzyme complex varies, depending on the relative abundance of components present at the site of aggregate formation.

Biochim Biophys Acta, 1997 Oct 18, 1348(3), 273 - 86
Diacylglycerol partitioning and mixing in detergent micelles: relevance to enzyme kinetics; Zhou C et al.; For many of the enzymes that utilize or produce diacylglycerols, detergent mixed micelles are often used in assay systems to solubilize the lipophilic substrates or products . The assumption is often made that the diacylglycerol (DAG) is solubilized and well mixed throughout the population of micelles during the time course of the assay . In the present work the partitioning and exchange dynamics of diacylglycerols (from dihexanoyl-DAG to didecanoyl-DAG) in a variety of detergent micelles have been studied by NMR and fluorescence methods . In all detergents, the longer the DAG chain lengths, the more detergent is required for solubilization . However, efficiency of solubilization varies tremendously with Triton X-100 the most efficient (i.e . the least detergent is required), and deoxycholate the least efficient in solubilizing DAG . The mixing and exchange dynamics of pyrene-labeled DAG molecules in these micelles (measured by stopped-flow fluorescence) were fastest for Triton X-100 and slowest with charged bile salt micelles . Of the detergent systems characterized, Triton X-100 appears to be the optimal detergent for use in assays of enzymes that interact with DAG (beta-octylglucoside and diheptanoylphosphatidylcholine have good exchange dynamics, but higher amounts of these detergents are needed to solubilize DAG) . Bile salt micelles provide the least solubilization and the slowest exchange kinetics (so slow that this could be a significant problem in some enzyme assays) . This information on DAG behavior in micelles is discussed with respect to assays of an enzyme that generates DAG as product (phospholipase C) and one that uses DAG as substrate (DAG kinase) . Although slow exchange of DAG occurs in some micelle systems, this does not appear to be a rate-limiting step in the kinetics for either of these enzymes.

Nippon Ika Daigaku Zasshi, 1997 Oct, 64(5), 422 - 7
Effects of maternal infection on prostaglandin production and uterine contraction in late-gestation pregnant goats; Tanaka K et al.; The main objective of this study was to evaluate the relationship between maternal infection induced by pyrogen administration and the changes in maternal and fetal plasma levels of prostaglandin E2 (PGE2) and F 2 alpha (PGF 2 alpha) and uterine contraction . We administered one mg of pyrogen (E . coli endotoxin) into the maternal femoral vein; then then the changes in maternal and fetal core temperatures, plasma levels of PGE2, {PGE2} and PGF 2 alpha, {PGF 2 alpha} . pO2, pCO2, pH and maternal uterine contractions were measured in late-gestation pregnant goats (n = 8) . Maternal and fetal core temperatures were elevated significantly 30 min after pyrogen administration (p < 0.05), reached their peak levels after 3 hours, and after 5 hours mean core temperatures returned to levels indistinguishable from initial control . Maternal plasma {PGE2} and {PGF 2 alpha} increased to 4318 +/- 321 pg/ml after 1 hour and to 670.8 +/- 58.4 pg/ml after 3 hours respectively (p < 0.05) . Fetal plasma {PGF 2 alpha} increased significantly (p < 0.05) to 811.0 +/- 64.0 pg/ml after 1 hour; however, plasma {PGE2} did not change throughout the study . Periodic uterine contractions appeared after 3 hours and disappeared after 5 hours . These results suggest that pyrogen induces the elevation of maternal and fetal core temperatures and prostaglandin production, and that eventually causes uterine contractions.

Comp Biochem Physiol A Physiol, 1997 Oct, 118(2), 291 - 5
Stimulation of three distinct guanylate cyclases induces mucosal surface alkalinisation in rat small intestine in vitro; Fawcus K et al.; The absorptive surface of the small intestine is isolated from bulk pH changes in the luminal contents by a zone of maintained low pH, the acid microclimate . The present study set out to compare the effects of stimulation of each of the three guanylate cyclases (GCs) expressed in the intestinal mucosa on the pH microclimate of rat jejunum in vitro . The tissue was exposed to specific ligands for each of the GCs and mucosal surface pH determinations were made by a miniaturised glass pH electrode . The ligands used were E . coli STa enterotoxin, atrial natriuretic peptide (ANP) and nitric oxide (NO, via the donor sodium nitroprusside (SNP)) . Challenge from all three agonists resulted in significant alkalinisation of the jejunal mucosa . The actions of SNP were blocked by the soluble GC inhibitor, methylene blue (MB) whereas those of STa were unaffected by MB . The data are consistent with previous observations that cGMP-induced inhibition of brush border Na+/H+ exchange results in elevation of mucosal surface pH . We conclude that all three of the identified GC pathways in the intestinal mucosa are capable of contributing to the control of mucosal acidification in the upper small intestine.

J Biol Chem, 1997 Nov 14, 272(46), 29356 - 63
Molecular cloning and expression of rat liver endo-alpha-mannosidase, an N-linked oligosaccharide processing enzyme; Spiro MJ et al.; A clone containing the open reading frame of endo-alpha-D-mannosidase, an enzyme involved in early N-linked oligosaccharide processing, has been isolated from a rat liver lambdagt11 cDNA library . This was accomplished by a strategy that involved purification of the endomannosidase from rat liver Golgi by ligand affinity chromatography (Hiraizumi, S., Spohr, U., and Spiro, R . G . (1994) J . Biol . Chem . 269, 4697-4700) and preparative electrophoresis, followed by sequence determinations of tryptic peptides . Using degenerate primers based on these sequences, the polymerase chain reaction with rat liver cDNA as a template yielded a 470-base pair product suitable for library screening as well as Northern blot hybridization . EcoRI digestion of the purified lambda DNA released a 5.4-kilobase fragment that was amplified in Bluescript II SK(-) vector . Sequence analysis indicated that the deduced open reading frame of the endomannosidase extended from nucleotides 89 to 1441, encoding a protein of 451 amino acids and corresponding to a molecular mass of 52 kDa . Data base searches revealed no homology with any other known protein . When a vector coding for this protein fused to an NH2-terminal peptide containing a polyhistidine region was introduced into Escherichia coli, high levels of the enzyme were expressed upon induction with isopropyl-beta-D-thiogalactoside . Purification of the endomannosidase to electrophoretic homogeneity from E . coli lysates was accomplished by Ni2+-chelate and Glcalpha1-->3Man-O-(CH2)8CONH-Affi-Gel ligand chromatographies . Polyclonal antibodies raised against this protein reacted with Golgi endomannosidase . By both immunoblotting and silver staining, the purified E . coli-expressed enzyme was approximately 8 kDa smaller than anticipated from the open reading frame; timed induction studies indicated that this was due to scission of the enzyme's COOH-terminal end by host cell proteases . All rat tissues examined demonstrated mRNA levels (4.9-kilobase message) for the endomannosidase that correlated well with their enzyme activity.

J Biol Chem, 1997 Nov 14, 272(46), 29255 - 62
The smallest carbamoyl-phosphate synthetase . A single catalytic subdomain catalyzes all three partial reactions; Guy HI et al.; Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit . The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis . Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively . Previous studies of individually cloned CPS.A and CPS . B from E . coli and mammalian CPSase have shown that homologous dimers of either of these "half-molecules" could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis . Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B . When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein . In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14-16-fold . The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.

J Biol Chem, 1997 Nov 14, 272(46), 28999 - 9004
Construction of chimeric enzymes out of maize endosperm branching enzymes I and II: activity and properties; Kuriki T et al.; Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties, and to elucidate the domain(s) that determines the differences, chimeric genes consisting of part mBE I and part mBE II were constructed . When expressed under the control of the T7 promoter in Escherichia coli, several of the chimeric enzymes were inactive . The only fully active chimeric enzyme was mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was exchanged for that of mBE I at a BspHI restriction site and was purified to homogeneity and characterized . Another chimeric enzyme, mBE I-II HindIII, in which the amino-terminal end of mBE II was replaced with that of mBE I, had very little activity and was only partially characterized . The purified mBE II-I BspHI exhibited higher activity than wild-type mBE I and mBE II when assayed by the phosphorylase a stimulation assay . mBE II-I BspHI had substrate specificity (preference for amylose rather than amylopectin) and catalytic capacity similar to mBE I, despite the fact that only the carboxyl terminus was from mBE I, suggesting that the carboxyl terminus may be involved in determining substrate specificity and catalytic capacity . In chain transfer experiments, mBE II-I BspHI transferred more short chains (with a degree of polymerization of around 6) in a fashion similar to mBE II . In contrast, mBE I-II HindIII transferred more long chains (with a degree of polymerization of around 11-12), similar to mBE I, suggesting that the amino terminus of mBEs may play a role in the size of oligosaccharide chain transferred . This study challenges the notion that the catalytic centers for branching enzymes are exclusively located in the central portion of the enzyme; it suggests instead that the amino and carboxyl termini may also be involved in determining substrate preference, catalytic capacity, and chain length transfer.

J Biol Chem, 1997 Nov 14, 272(46), 28994 - 8
Kinetic partitioning . Poising SecB to favor association with a rapidly folding ligand; Diamond DL et al.; Chaperones are a class of proteins that possess the remarkable ability to selectively bind polypeptides that are in a nonnative state . The selectivity of SecB, a molecular chaperone in Escherichia coli, for its ligands can be explained in part by a kinetic partitioning between folding of the polypeptide and association with SecB . It has clearly been established that kinetic partitioning can be poised to favor association with SecB by changing the rate constant for folding of the ligand . We now demonstrate that binding to SecB can be given a kinetic advantage over the pathway for folding by modulating the properties of the chaperone . By poising SecB to expose a hydrophobic patch, we were able to detect a complex between SecB and maltose-binding protein under conditions in which rapid folding of the polypeptide otherwise precludes formation of a kinetically stable complex . The data presented here are interpreted within the framework of a kinetic partitioning between binding to SecB and folding of the polypeptide . We propose that exposure of a hydrophobic patch on SecB increases the surface area for binding and thereby increases the rate constant for association . In this way association of SecB with the polypeptide ligand has a kinetic advantage over the pathway for folding.

J Biol Chem, 1997 Nov 14, 272(46), 28980 - 8
Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli; Apostol I et al.; We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli . The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing . It appears that substitution is distributed across both the beta- and di-alpha-globins in purified recombinant hemoglobin . The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture . This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNALeu, similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E . coli.

J Biol Chem, 1997 Nov 14, 272(46), 28906 - 11
Anti-mutagenic activity of DNA damage-binding proteins mediated by direct inhibition of translesion replication; Paz-Elizur T et al.; DNA lesions that block replication can be bypassed in Escherichia coli by a special DNA synthesis process termed translesion replication . This process is mutagenic due to the miscoding nature of the DNA lesions . We report that the repair enzyme formamido-pyrimidine DNA glycosylase and the general DNA damage recognition protein UvrA each inhibit specifically translesion replication through an abasic site analog by purified DNA polymerases I and II, and DNA polymerase III (alpha subunit) from E . coli . In vivo experiments suggest that a similar inhibitory mechanism prevents at least 70% of the mutations caused by ultraviolet light DNA lesions in E . coli . These results suggest that DNA damage-binding proteins regulate mutagenesis by a novel mechanism that involves direct inhibition of translesion replication . This mechanism provides anti-mutagenic defense against DNA lesions that have escaped DNA repair.

J Steroid Biochem Mol Biol, 1997 Apr, 61(3-6), 117 - 26
Diverse function of aromatase and the N-terminal sequence deleted form; Osawa Y et al.; The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated . Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli . Aromatase cytochrome P450 was reconstituted and incubated with {6alpha,7alpha-(3)H2,4-(14)C}estradiol, 7-ethoxycoumarin, and {N-methyl-(3)H3}cocaine . 6Alpha-hydroxy{7alpha-(3)H,4-(14)C}estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established . The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1) . Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase . The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1) . The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1) . All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2 . The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

Thromb Haemost, 1997 Oct, 78(4), 1209 - 14
Effects of plasma kallikrein specific inhibitor and active-site blocked factor VIIa on the pulmonary vascular injury induced by endotoxin in rats; Uchiba M et al.; The acute respiratory distress syndrome (ARDS) is a serious complication of sepsis . To evaluate the role of the coagulation system in the pathogenesis of ARDS in sepsis, we examined the effects of the administration of a synthetic plasma kallikrein specific inhibitor (PKSI) and of active-site blocked factor VIIa (DEGR-VIIa) on the pulmonary vascular injury induced by E . coli endotoxin (ET) in rats . Administration of PKSI prevented the pulmonary vascular injury induced by ET as well as pulmonary histological changes in animals administered ET, but it did not affect the intravascular coagulation . The opposite effect was seen with DEGR-VIIa, which prevented the intravascular coagulation but not the pulmonary vascular injury . PKSI did not inhibit the activation of the complement system induced by ET leading to the activation of neutrophils . Findings suggest that PKSI may prevent the pulmonary vascular injury induced by ET by inhibiting kallikrein, which activates the neutrophils . The intrinsic pathway of coagulation may be more important than the extrinsic pathway in the pulmonary vascular injury produced by ET.

Mol Biochem Parasitol, 1997 Nov, 89(2), 271 - 82
Identification and characterization of SRS1, a Toxoplasma gondii surface antigen upstream of and related to SAG1; Hehl A et al.; Previous investigations of the major surface antigen (SAG1) promoter of Toxoplasma gondii indicated an ability to function bi-directionally in transient transformation assays at least . This suggests there might be another tachyzoite-specific gene being divergently transcribed from the SAG1 promoter in its normal chromosomal location . To investigate this possibility we have characterized the region upstream of SAG1 and report here a co-directional transcription unit coding for a probable GPI-anchored surface protein with homology to SAG1 and SAG3 . This antigen, which had not previously been identified in surface iodination experiments is given the acronym SRS1, for SAG1-related sequence 1 . Genomic organization and sequence of a full-length cDNA of SRS1 are presented . Antisera against a recombinant SRS1 protein produced in Escherichia coli, recognize a specific band of 46 kDa in parasite lysates which corresponds to the largest of the GPI-anchored proteins by Western blot . The possible role of this previously unidentified surface antigen is discussed.

Mol Biochem Parasitol, 1997 Nov, 89(2), 195 - 207
Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines; Lilley CJ et al.; Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines . The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H . glycines using consensus oligonucleotide primers . Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids . One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity . HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences . Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues . The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli . Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI . HGSP-III showed no activity against the synthetic substrates tested . The information gained from these studies indicates that serine proteinases are an important group of enzymes in H . glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.

Mol Microbiol, 1997 Sep, 25(5), 933 - 44
Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway; Clarke DJ et al.; DjIA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N-terminus . The overproduction of DjIA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two-component signal transduction pathway . We now show that both the TMD and the J-region are essential for the induction of cps expression observed with the overproduction of DjIA . Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjIA . These mutations were shown not to affect the localization, stability or topology of the mutant DjIA proteins . We propose that these mutations are affecting specific interactions between the TMD of DjIA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

Mol Microbiol, 1997 Sep, 25(5), 913 - 31
Positive control of the two-component RcsC/B signal transduction network by DjlA: a member of the DnaJ family of molecular chaperones in Escherichia coli; Kelley WL et al.; The membrane-anchored DjIA protein represents the third member of the DnaJ 'J-domain' family of Escherichia coli that includes DnaJ and CbpA . DjIA possesses a J-domain at its extreme C-terminus but shares no additional homology with DnaJ . Our genetic analysis suggests that DjIA acts in concert with the RcsB/C two-component signal transduction system to augment induction of the cps (capsular polysaccharide) operon and synthesis of colanic acid mucoid capsule . The DjIA J-domain is essential for the observed stimulation of this pathway as deletion, or introduction of the mutation H233Q, within the highly conserved HPD tripeptide abolished all inducing activity . Deletion of the transmembrane anchor sequence also abolished all inducing activity . djIA is not an essential gene under all conditions tested, nor is it essential for mucoid capsule biosynthesis; however, strong overexpression leads to rapid loss of cell viability suggesting that the gene is normally tightly regulated . Northern analysis revealed that djIA message was extremely unstable but could be induced or stabilized in response to cold shock . The activation of the cps operon by DjIA is dependent upon both DnaK(Hsp70) and GrpE, and therefore we propose a role for DjIA, together with this chaperone machine, as a novel regulator of a two-component histidine kinase signal transduction pathway.

Mol Microbiol, 1997 Sep, 25(5), 883 - 91
Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase; Beard SJ et al.; A transposon (Tn 10dCam) insertion mutant of Escherichia coli K-12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II) . The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance) . The metal-sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90-3684.60 kb on the physical map . Insertion of a kanamycin resistance (KnR) cassette at a SalI site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)-sensitive phenotype . DNA sequence analysis revealed that the KnR cassette was located within the putative promoter region of an ORF (o732 or yhhO) predicted to encode a protein of 732 amino acids, similar to cation transport P-type ATPases in the Cpx-type family . Inverse PCR and sequence analysis revealed that the Tn 10dCam element was located within o732 in the genome of the zinc(II)-sensitive mutant . The zntA mutant had elevated amounts of intracellular and cell surface-bound Zn(II), consistent with the view that zntA+ encodes a zinc(II) efflux protein . Exposure of the zntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface-bound metal ions.

Mol Microbiol, 1997 Sep, 25(5), 871 - 82
The Pai-associated leuX specific tRNA5(Leu) affects type 1 fimbriation in pathogenic Escherichia coli by control of FimB recombinase expression; Ritter A et al.; The uropathogenic Escherichia coli strain 536 (06:K15:H31) carries two large chromosomal pathogenicity islands (Pais) . Both Pais are flanked by tRNA genes . Spontaneous deletion of Pai II results in truncation of the leuX tRNA5Leu gene . This tRNA is required for the expression of type 1 fimbriae (Fim) and other virulence factors . Transcription of fimA, encoding the major type 1 fimbrial subunit is controlled by an invertable DNA switch . The inversion is catalysed by two recombinases, FimB and FimE . FimB is able to turn the switch on, FimE only off . The fimB gene of strain 536 contains five TTG codons recognized by tRNA5Leu, fimE contains only two . It was proposed that turning on the fim switch requires efficient translation of FimB, in turn requiring tRNA5Leu . Strains in which the TTG codons in fimB were replaced with CTG codons at the wild-type locus were able to produce type 1 fimbriae in the absence of leuX . fimB transcription was influenced by the presence of leuX, but only slightly affected by the presence or absence of leuX codons in fimB . FimB translation was significantly higher from codon-replaced fimB genes than that of wild-type fimB genes in various strain backgrounds . The fim switch was shown to be switched off in leuX-derivatives of E . coli 536, but could be found in the on position when the codon-altered fimB gene was exchanged into the chromosome of these strains . From these data, it is apparent that tRNA5Leu is required for efficient translation of FimB, in turn, leading to type 1 fimbrial expression.

Mol Biol Evol, 1997 Nov, 14(11), 1125 - 31
A graphical method for detecting recombination in phylogenetic data sets; McGuire G et al.; Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence . The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites . The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis . A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here . This method locates putative recombination breakpoints by moving a window along the sequence . The performance of the method is assessed by simulation and by its application to a real data set.

Vaccine, 1997 Oct, 15(15), 1598 - 605
Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS in comparison with Porphyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A (compound 506); Ogawa T et al.; Helicobacter pylori lipid A, characterised by a glucosamine beta (1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-positions, respectively, exhibited no or very low endotoxic activities, i.e . lethal toxicity in galactosamine-loaded mice, pyrogenicity for rabbits and the activity of the Limulus test compared with Escherichia coli-type synthetic lipid A (compound 506), which possesses beta-(1-6)-linked glucosamine disaccharide 1,4'-bisphosphate, with two acyloxyacyl groups at the 2'- and 3'-positions and two 3-hydroxytetradecanoyl groups at the 2- and 3-positions . The endotoxic properties of H . pylori lipid A were also a little weaker than those of the low endotoxic lipid A of P . gingivalis, which has 1-phospho beta-(1-6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2'-positions, respectively . Further, the mitogenic activity of H . pylori lipid A in murine splenic mononuclear cells was also less than those of P . gingivalis lipid A and compound 506 . However, H . pylori lipid A induced comparable production of interleukin-6 (IL-6) by human peripheral blood mononuclear cells (PBMC) compared with P . gingivalis lipid A and compound 506 . H . pylori lipid A also increased human natural killer cell activity, and strongly agglutinated rabbit erythrocytes . However, the lipid As of H . pylori and P . gingivalis showed lower activities in inducing tumour necrosis factor alpha (TNF-alpha) production by human PBMC and IL-8 production by human gingival fibroblasts than that of compound 506 . The structural feature of H . pylori lipid A may be associated with low endotoxic properties and potent immunobiological activities.

Vaccine, 1997 Nov, 15(16), 1784 - 90
Effects of frequent intranasal administration of adjuvant-combined influenza vaccine on the protection against virus infection; Tamura S et al.; In previous papers, we have shown that Escherichia coli heat-labile enterotoxin B subunit, supplemented with a trace amount of the holotoxin (LTB*) could be used as a potent adjuvant for a nasal influenza HA (haemagglutinin) vaccine in humans . The present study was designed to determine whether the effectiveness of a combined LTB*-HA vaccine could be limited by preexisting immunity to LTB and how many times the adjuvant-combined vaccine could be administered intranasally without reducing its protective efficacy in BALB/c, C3H and B10 mice . The magnitude of both nasal and serum Ab responses to HA vaccine was correlated with the degree of protection against virus infection . Higher doses of LTB*-combined vaccine were required for inducing high enough levels of anti-HA Ab responses to provide complete protection in low responder mice . Repeated pretreatments with LTB* alone (more than six times), which provided high levels of preexisting Abs to LTB, inhibited the induction of anti-HA Ab responses and reduced the protective efficacy of the adjuvant-combined vaccine . However, the LTB*-combined vaccine could be given repeatedly (about ten times) to mice without reducing the effectiveness of the adjuvant-combined vaccine . These results suggest that the LTB*-combined nasal influenza vaccine can be given to humans once every few years when an epidemic of influenza may occur.

Vaccine, 1997 Nov, 15(16), 1741 - 7
Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi; Seong SY et al.; Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells . However, O . tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified . The authors immunized mice and rabbits with the recombinant 56 kDa protein of O . tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O . tsutsugamushi attachment to or penetration of L929 cells . O . tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA) . O . tsutsugamushi growth in L929 cells was determined by {3H}thymidine uptake assay . By IFA, we observed a 96% reduction of attachment or penetration of O . tsutsugamushi treated with rabbit anti-MBP-Bor56 sera . {3H}thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O . tsutsugamushi growth, when compared to mouse anti-MBP sera . These results suggest that the 56 kDa protein of O . tsutsugamushi plays an important role in O . tsutsugamushi attachment to or penetration of cells.

J Pharm Pharmacol, 1997 Oct, 49(10), 988 - 90
Nitric oxide modulates the acute increase of gastrointestinal transit induced by endotoxin in rats: a possible role for tachykinins; Martinez-Cuesta MA et al.; Because of the evidence that endogenous nitric oxide (NO) plays an essential role in the physiological regulation of gastrointestinal motility we have investigated, by use of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), the role of endogenous NO in the acute endotoxin-induced changes of gastrointestinal transit . Pre-treatment with E . coli endotoxin (100 micrograms kg-1, i.v.) induced a significant increase in the gastrointestinal transit of a charcoal suspension in anaesthetized rats . Previous administration of the NO synthase inhibitor, L-NAME (10 mg kg-1, i.v.) significantly prevented the effects of endotoxin . L-arginine (200 mg kg-1, i.v.) and the substance P antagonist {D-Pro2, D-Trp7,9}-substance P (SPA), significantly reversed the effects of L-NAME on gastrointestinal transit in rats treated with endotoxin . Pre-treatment with dexamethasone (5 mg kg-1, s.c., twice), an inhibitor of the expression of inducible NO synthase, did not affect the increase in the gastrointestinal transit through constitutive NO synthesis . The results suggest that constitutive nitric oxide is involved in the increase of gastrointestinal transit induced by endotoxin and that the reduction in transit induced by L-NAME in endotoxin-treated rats is mediated by endogenous tachykinins.

J Am Vet Med Assoc, 1997 Nov 1, 211(9), 1155 - 7
Use of full cortical allograft to repair a metatarsal fracture in a foal; Cassotis NJ et al.; A 4-month-old Quarter Horse colt was admitted for repair of an open, comminuted fracture of the proximal portions of the diaphyses of the left second, third, and fourth metatarsal bones . Initial repair included internal fixation and cancellous bone graft . However, the third metatarsal bone became infected and failed to heal . After removal of infected portions of the bone, a 5-cm, fullthickness cortical allograft was placed in the defect . Rigid internal fixation provided stability for the allograft and remaining fracture fragments so that the horse was able to bear weight on the second and fourth metatarsal bones . The allograft was ultimately resorbed; however, appositional bone growth permitted a massive, functional metatarsal bone to form that incorporated the second, third, and fourth metatarsal bones . The colt went on to compete successfully, long term, as a show horse.

Cell, 1997 Oct 31, 91(3), 347 - 56
Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps; Webb BL et al.; In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated . RecR protein alone has no effect, and RecF protein alone has a reduced activity . The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered . A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP . These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair.

Int Arch Allergy Immunol, 1997 Nov, 114(3), 293 - 7
Protection against verocytotoxin in mice induced by liposome-coupled verocytotoxin; Naito S et al.; Purified verocytotoxins (VTs), VT1 and VT2, were coupled to liposomes via glutaraldehyde . During the coupling procedure, both VT1 and VT2 were detoxified . Intraperitoneal injection in BALB/c mice with either VT1-liposome or VT2-liposome induced a substantial amount of anti-VT1 or anti-VT2 IgG antibody production, respectively . Mice immunized with VT2-liposome were protected against intravenous challenge with a lethal dose of VT2 and the degree of protection correlated well with the amount of IgG induced against VT2 . Although VT1-liposome failed to induce protection against VT1, the decrease of the body weight observed after the toxin challenge correlated inversely with the amount of anti-VT1 IgG induced, suggesting that VT1 neutralizing antibody was present in VT1-liposome-immune mice . In addition, VT-liposome conjugate induced no detectable anti-VT IgE antibody production . These results demonstrate the potential ability of VT-liposome conjugates for the production of VT vaccine which induces protection against VTs.

Int Arch Allergy Immunol, 1997 Nov, 114(3), 229 - 36
Molecular cloning and expression of cynomolgus monkey interferon-gamma cDNA; Tatsumi M et al.; Since the discovery of its involvement in the pathogenesis of simian immunodeficiency virus infection, the role of macaque monkey interferon-gamma (IFNgamma) has been a focus of particular interest . The purpose of this study was to express bioactive recombinant monkey IFNgamma, as well as clone cynomolgus monkey IFNgamma cDNA . The isolation of a cDNA encoding cynomolgus monkey IFNgamma was performed using a reverse transcriptase polymerase chain reaction-based strategy with activated monkey lymphocyte cDNA as templates . Cynomolgus monkey IFNgamma consists of 165 amino acids including a putative signal sequence and has 94, 60 and 39% identity to human, bovine and murine homolgues, respectively . Monkey IFNgamma cDNA was highly expressed as three distinct secreted proteins by a recombinant baculovirus system . The secreted proteins were revealed to have antiviral activity up to 10(8) units/ml characteristic of authentic IFNgamma by a bioassay of a cytopathic effect reduction assay.

Eur J Biochem, 1997 Oct 1, 249(1), 318 - 24
The TATA-box-binding protein (TBP) of Halobacterium salinarum . Cloning of the tbp gene, heterologous production of TBP and folding of TBP into a native conformation; Soppa J et al.; The TATA-box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eucarya and Archaea . Using a tbp-specific probe, a 4.5-kbp genomic fragment from Halobacterium salinarum was cloned and sequenced . It contained the tbp gene and the 5'-ends of two additional open reading frames, but surprisingly, 70% of the cloned fragment (3.2 kbp) was devoid of coding capacity or similarity to database sequences . The deduced halobacterial TBP exhibits sequence similarities to other archaeal (41-43%) as well as to eucaryal (27-38%) TBP . A comparative analysis showed that the archaeal and eucaryal TBP form two related monophylic protein families, and the archaeal TBP possess features which separate them from eucaryal TBP . Compared with the other TBP, the halobacterial TBP is unique in having a high excess of negatively charged residues . A histidine-tagged version of the halobacterial TBP was produced in Escherichia coli in a denatured conformation and purified by means of Ni-chelating chromatography . CD spectroscopy was used to monitor TBP secondary structure and the conditions necessary for folding it into a native conformation . In the absence of denaturating agents, the folded as well as the unfolded state were found to be stable over a wide range of salt concentrations . Properly folded TBP was shown to bind to a halobacterial TATA-box-containing DNA fragment, indicating that the fusion protein can be used to characterize DNA recognition by the halobacterial TBP.

Eur J Biochem, 1997 Oct 1, 249(1), 293 - 300
Guanine-nucleotide binding and hydrolyzing kinetics of ORrab2, a rice small GTP-binding protein expressed in Escherichia coli; Seo HS et al.; The ORrab2 gene encodes a GTP-binding protein of 23.169 kDa . The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP-binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%), Hrab4 (51.9%), Hrab1 (46.2%), YPT (40.7%), Hrab3B (40.0%), Hrab3A (38.1%), SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%) . To analyze the biochemical properties of this protein, an ORrab2 cDNA was overexpressed in Escherichia coli and the protein purified by Ni2+-nitrilotriacetic acid agarose and hydroxyapatite column chromatography . The molecular mass of the protein bearing a His-tag is approximately 28.2 kDa . The guanine-nucleotide binding and hydrolyzing activity of ORrab2 increased with non-ionic C12E10 (polyoxyethylene 10-lauryl ether) and ionic Chaps detergent treatment . ORrab2 bound maximally 1.03 mol of {gamma-35S}GTP{S}/mol of protein with a Kd value of 56.83 nM . The ratios k(off GDP)/k(off GTP) of ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP . The rate (k(cat)) of Pi release against {gamma-32P}GTP bound ORrab2 in a steady state and the rate of hydrolysis of {gamma-32P}GTP (kGTPase) were calculated to be 432 x 10(-4) +/- 8 x 10(-4) min(-1) and 172 x 10(-4) +/- 2 x 10(-4) min(-1), respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4.

Eur J Biochem, 1997 Oct 1, 249(1), 280 - 5
Methanol:coenzyme M methyltransferase from Methanosarcina barkeri . Zinc dependence and thermodynamics of the methanol:cob(I)alamin methyltransferase reaction; Sauer K et al.; In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M . This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2) . Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol {Sauer, K., Harms, U . & Thauer, R . K . (1997) Eur . J . Biochem . 243, 670-677} . We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli . MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol . MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3) . The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

Eur J Biochem, 1997 Oct 1, 249(1), 134 - 41
Cross-linking of chloroplast F0F1-ATPase subunit epsilon to gamma without effect on activity . Epsilon and gamma are parts of the rotor; Schulenberg B et al.; Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6-->Ser mutant of subunit epsilon of spinach chloroplast F0F1 ATP synthase . Wild-type and engineered epsilon were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1 lacking the epsilon subunit {F1(-epsilon)} . Cys-containing epsilon variants were modified with a sulfhydryl-reactive photolabile cross-linker . Photocross-linking of epsilon to F1(-epsilon) yielded the same SDS gel pattern of cross-link products independent of the presence or absence of Mg2+ x ADP, phosphate and Mg2+ x ATP . Epsilon (wild type) {Ser6,Cys28}epsilon and {Ser6,Cys41}epsilon were cross-linked with subunit gamma . With chloroplast F0F1 the same cross-link pattern was obtained, except for one extra cross-link, probably between {Ser6,Cys28}epsilon and F0 subunit III . {Ser6,Cys17}epsilon and {Ser6,Cys85}epsilon did not produce cross-links . Cross-linking of epsilon, {Ser6,Cys28}epsilon, {Ser6,Cys41}epsilon to gamma in soluble chloroplast F1 impaired the ability of epsilon to inhibit Ca2+-ATPase activity . The Mg2+-ATPase activity of soluble F1 (measured in the presence of 30% MeOH) was not affected by cross-linking epsilon with gamma . Functional reconstitution of photophosphorylation in F1-depleted thylakoids was observed with F1 in which gamma was cross-linked to {Ser6,Cys28}epsilon or {Ser6,Cys41}epsilon but not with wild-type epsilon . In view of the intersubunit rotation of gamma relative to (alphabeta)3, which is driven by ATP hydrolysis, gamma and epsilon would seem to act concertedly as parts of the 'rotor' relative to the 'stator' (alphabeta)3.

Int J Biochem Cell Biol, 1997 Apr, 29(4), 659 - 66
Unusual effect of clusters of rare arginine (AGG) codons on the expression of human interferon alpha 1 gene in Escherichia coli; Ivanov IG et al.; The human interferon (hIFN alpha 1) gene contains 11 arginine (Arg) codons AGG or AGA, which are extremely rare for bacteria, four of which are organized in tandems . The two AGG tandems (corresponding to Arg12 Arg13 and Arg163 Arg164) are known to inhibit the translation of hIFN alpha 1 mRNA and therefore they are considered to be responsible for the poor expression of hIFN alpha 1 gene in bacterial cells . To study the effect of these two tandems on the expression of hIFN alpha 1 in E . coli, four new gene variants were designed to contain preferential Arg codons (CGT) substituted for the rare AGG codons in either the first, the second or both AGG tandems . We found that, whereas the yield of hIFN alpha 1 protein per cell remained unchanged, the level of hIFN alpha 1 mRNA decreased gradually (by a factor of two) with the consecutive substitution of the first, second and both AGG tandems . These results indicated, first, that the AGG clusters might have a stabilizing effect on the mRNA, and second, that mRNAs devoid of such clusters were translated at a higher rate in vivo . The protein products of the four genes (having the same amino acid sequence) showed different specific antiviral activity . The most active was the product of gene hIFN alpha 1(c) in which the second AGG tandem (corresponding to Arg163, Arg164) was preserved while the least active was the protein of gene hIFN alpha 1(d) (devoid of both AGG clusters) . The role of the AGG tandems in folding of the gene product is discussed.

Int J Biochem Cell Biol, 1997 Apr, 29(4), 589 - 94
Partial inactivation of chorismate mutase-prephenate dehydrogenase from Escherichia coli in the presence of analogues of chorismate; Christopherson RI; Chorismate-5,6-epoxide, chorismate-5,6-diol, various adamantane derivatives and 2-hydroxy-phenyl acetate are structural analogues of chorismate that act as competitive inhibitors of both the chorismate mutase and prephenate dehydrogenase activities of the bifunctional enzyme, hydroxyphenylpyruvate synthase . The interactions of these chorismate analogues with both activities of the synthase are investigated further . Chorismate mutase and prephenate dehydrogenase activities were assayed spectrophotometrically at 290 and 340 nm, respectively . Data were fit by non-linear regression to appropriate equations describing the time-dependent formation of product or decay of enzymic activity . In the presence of these chorismate analogues, both the mutase and dehydrogenase activities undergo a time-dependent partial inactivation . Progress curves for synthesis of product by the mutase or dehydrogenase in the presence of chorismate-5,6-epoxide, chorismate-5,6-diol or adamantane-1,3-diacetate resemble time-courses characteristic of slow-binding inhibition . However, if the bifunctional enzyme was preincubated with a chorismate analogue prior to addition of substrate, only a minor proportion of enzymic activity was recovered, excluding the possibility of reversible, slow-binding inhibition . When hydroxyphenylpyruvate synthase binds certain chorismate analogues to form an EI complex, there is a slow conformational transition to an ET complex, which may be susceptible to oxidation leading to partial inactivation . Some protection against this inactivation is provided by high concentrations of dithiothreitol (20 mM), suggesting that the inactivation may be due to chemical oxidation.

Glycobiology, 1997 Oct, 7(7), 955 - 64
Generation of constitutive and inducible trans-sialylation dominant-negative phenotypes in Trypanosoma brucei and Trypanosoma cruzi; Engstler M et al.; Trans-sialylation is a unique enzymatic process that is restricted to some trypanosome species . By expressing developmentally regulated trans-sialidases, these protozoan parasites cleave sialic acids from host glycoconjugates and transfer them to acceptors on their own cell surfaces . The biological function of this process is not understood, but trans-sialylation is expected to be important in the invasion of mammalian cells by Trypanosoma cruzi and the survival of Trypanosoma brucei within its insect vector . Since a conventional gene knockout approach was precluded, we developed a dominant-negative strategy, in which fusion proteins consisting of a bacterial sialidase and trypanosome proteins were expressed in T.brucei and T.cruzi . The strong recombinant sialidase activity shifted the reaction equilibrium from sialic acid transfer to hydrolysis, in this way creating a sialic-acid-negative phenotype . Taking advantage of a recently introduced inducible expression system, we were able to control the expression of sialidase fusion proteins in T.brucei . Reversion of the sialic-acid-negative state to wild-type sialylation was accomplished by selective inhibition of the foreign sialidase, leaving the parasite trans-sialidase unaffected . Both desialylation and resialylation of trypanosomes was rapidly achieved . Our results show that neither T.brucei nor T.cruzi require sialic acids for survival in vitro, ruling out the involvement of sialylation in cell surface integrity . The versatile system introduced here will allow a detailed in vivo study of the role of trans-sialylation during the trypanosome infection cycle . Furthermore, cell-surface sialic acids are implicated in a multitude of (patho-) biochemical processes in other organisms . The quantitative and qualitative manipulation of cell surface sialic acids, by expressing of counteracting enzymes, constitutes a novel approach with potentially broad applications in glycobiology.

Planta, 1997 Oct, 203(2), 237 - 44
A vegetative storage protein homolog is expressed in the growing shoot apex of hybrid poplar; Lawrence SD et al.; The ability of poplars (Populus deltoides Bartr . ex Marsh., and Populus trichocarpa Torr . and Gray) to sequester nitrogen in stems in preparation for winter has been associated with the massive accumulation of protein bodies in the bark and xylem ray parenchyma . These protein bodies contain a bark storage protein (BSP) that can account for up to 30% of the total soluble bark protein during the winter months . Perhaps the plant's ability to efficiently cycle nitrogen through BSP is an important aspect of its growth potential . Sequence analysis of BSP led to the identification of a leaf-associated homolog, win4, which was initially isolated because its transcript increased in abundance upon mechanical wounding . The goal of this work was to characterize this putative leaf-associated vegetative storage protein, and determine whether it might perform a storage role in vivo . Antibodies, produced against protein synthesized upon over-expression of the win4 coding region in Escherichia coli, were used to examine the relative abundance of WIN4 protein in response to supplemental nitrogen, and during development . The transcript and protein were most abundant in the youngest leaves and also increased with nitrogen fertilization . Immunolocalization of the protein was performed and showed that WIN4 was associated with cells surrounding the vasculature, and cells of the lower epidermis and stipules of immature leaves . Under moderate nitrogen fertilization regimes, WIN4 accounted for only about 2% of total soluble leaf protein; however, given the cellular specificity and enhancement with nitrogen, the protein is regulated in a manner similar to other vegetative storage proteins . Since poplar is amenable to DNA transformation and regeneration, it is now possible to ask direct questions about the role these proteins play in nitrogen storage in rapidly expanding or in dormant tissue . This type of analysis could determine whether these proteins mainly ameliorate the toxic effects of excess nitrogen, if they are instrumental in controlling nitrogen allocation or if they simply represent an efficient method for sequestering this valuable nutrient.

Development, 1997 Dec, 124(24), 4971 - 82
Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development; Yin Z et al.; The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells . These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors . Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts . We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation . This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo . Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression . We further show that the 180 bp enhancer also includes negatively acting sequences . Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm . In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes . We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area . Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.

J Vet Med Sci, 1997 Oct, 59(10), 917 - 21
Effect of oral egg antibody in experimental F18+ Escherichia coli infection in weaned pigs; Yokoyama H et al.; The protection conferred by egg antibody specific for F18-fimbriae against infection with F18+ Escherichia coli was studied in controlled passive immunization trials involving weaned pigs . Parameters of protection consisted of body weight gain, frequency and severity of diarrhea and recovery of the challenge strain of F18+ E . coli . Weaned pigs at four weeks of age were challenge exposed once daily for three days by oral inoculation with 10(11) cfu of virulent F18+ E . coli followed by daily administration of antibody supplemented feed for 9 days starting from the first challenge day 0 . Results showed a dose-dependent response to antibody treatment . The group of pigs given 1:50 titer of antibody in feed had less frequency of diarrhea (P < 0.01-0.05), higher rate of gain (P < 0.01) and lower isolation rate of challenge strain in rectal and intestinal swabs (P < 0.01) compared to non-treated control . In the same manner, the anti-F18 antibody significantly reduced adherence of F18+ E . coli to pig intestinal epithelial cells in vitro (P < 0.01) . Results suggest that egg antibodies specific for the F18 fimbriae is a suitable immunotherapeutic agent for pigs infected with F18+ E . coli and that pigs can be protected from overt clinical disease and the subsequent reduced performance arising from infection with this pathogen.

Zentralbl Bakteriol, 1997 Oct, 286(3), 383 - 8
Receptors on chicken erythrocytes for F42 fimbriae of Escherichia coli isolated from pigs; Leite Dda S et al.; Haemagglutination of purified F42 fimbriae was found to be inhibited by N-acetyl-galactosamine . Purified F42 fimbrial adhesin reacted with distinct membrane components from chicken erythrocytes (35, 37 and 40 kDa) in immunoblot analysis, suggesting that the binding occurred to proteins or glycoproteins.

J Vet Med Sci, 1997 Oct, 59(10), 927 - 9
Circulating tumor necrosis factor and interleukin-1 after administration of LPS in adult cows; Ohtsuka H et al.; This study examined the levels of serum tumor necrosis factor (TNF), interleukin-1 (IL-1) and plasma cortisol levels in adult cows after intravenous administration of lipopolysaccharide (LPS) extracted from Escherichia coli . The adult cows exhibited clinical signs of endotoxic shock . The mean concentrations of serum TNF was 557.9 U/ml and serum IL-1 was 90.6 U/ml at 2 hr after LPS administration following severe endotoxic shock . Plasma cortisol concentrations increased immediately and a peak level was 131.4 ng/ml at 1 hr after LPS challenge . These results suggested that circulating concentrations of TNF, IL-1, and cortisol may be associated with clinical signs of endotoxemia in adult cows.

Am J Vet Res, 1997 Nov, 58(11), 1291 - 9
Effect of pentoxifylline, flunixin meglumine, and their combination on a model of endotoxemia in horses; Baskett A et al.; OBJECTIVE: To compare effects of a single dose of pentoxifylline (PTX), flunixin meglumine (FM), and their combination (FM/PTX) in a model of equine endotoxemia . ANIMALS: 24 healthy horses, aged 2 to 15 years . PROCEDURE: 4 groups (n = 6/group) received 30 ng of Escherichia coli O55:B5 endotoxin/kg of body weight, i.v., over 30 minutes, and 1 of the following preparations 15 minutes before and 8 hours after endotoxin infusion: FM, 1.1 mg/kg; PTX, 8 mg/kg; FM/PTX, 1.1 mg of FM and 8 mg of PTX/kg; and saline solution bolus (ENDO) . Clinical and hematologic variables were measured over 24 hours . RESULTS: Compared with ENDO, FM given before endotoxin significantly reduced TxB2, and 6-keto-PGF1 concentrations, pulse, rectal temperature, and attitude score . Pentoxifylline given before endotoxin resulted in significantly higher 6-keto-PGF1 concentration at 1.5 hours and significantly lower PAI-1 activity at 12 hours . Tumor necrosis factor and IL-6 activities in horses given PTX alone were not significantly different from values in those given the saline bolus . FM/PTX induced effects similar to those of FM alone on endotoxin-induced changes in temperature and TxB2 concentration, and 6-keto-PGF1 concentration was significantly lower than that in horses of the ENDO group at 1 hour . In horses of the FM group, 6-keto-PGF1 concentration was significantly lower than that in horses of the ENDO group, from 0.5 hour to 2 hours . Horses of the FM and FM/PTX groups had significantly higher IL-6 activity at 1.5 and 2 hours than did horses of the PTX and ENDO groups; those of the FM and FM/PTX groups had significantly lower WBC count than did those of the PTX and ENDO groups . CONCLUSIONS: FM/PTX may help offset deleterious hemodynamic effects of endotoxin more effectively than does either FM or PTX alone.

Appl Environ Microbiol, 1997 Nov, 63(11), 4504 - 10
Characterization of DNA polymerase from Pyrococcus sp . strain KOD1 and its application to PCR; Takagi M et al.; The DNA polymerase gene from the archaeon Pyrococcus sp . strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no . D29671) . Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases . The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized . 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase . These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

Appl Environ Microbiol, 1997 Nov, 63(11), 4204 - 9
Recovery of culturability of an HOCl-stressed population of Escherichia coli after incubation in phosphate buffer: resuscitation or regrowth?
Dukan S, Levi Y, Touati D.
An Escherichia coli population harvested in exponential phase at about 10(8) cells/ml was treated in phosphate buffer with HOCl at concentrations ranging from 0.4 to 1 mg/liter (7.7 to 19 microM) . The HOCl stress resulted in the appearance of three cell subpopulations: a majority of dead (nonrespiring) cells, a few culturable cells (10(2) to 10(4)), and about 10(7) viable but nonculturable cells . In the absence of any added exogenous nutrient, a culturable population could be recovered after 1 day of incubation in phosphate buffer, and such a population would reach a cell density close to 10% of the initial density of the stressed population, whatever the initial number of survivors . When a small number of untreated cells were mixed with the stressed population, growth of the untreated cells was observed, demonstrating that damaged cells provided nutrients . Similarly, a filtrate and a disrupted-cell filtrate of the stressed population supported growth of untreated cells with the same efficiency . The number of CFU (untreated or stressed) at plateau phase depended on the initial density of the stressed cells . Taken together, these results suggest that recovery in phosphate buffer of an HOCl-stressed population is in large part due to growth of a few culturable cells at the expense of damaged cells . However, comparison of the growth rates of the stressed culturable population and of untreated bacteria growing in filtrate showed significantly faster growth of the stressed cells, a fact not fully compatible with the hypothesis that recovery is only the simple growth of survivors . We suggest, therefore, that in addition to growth of the few culturable stressed cells, there is repair and growth of some mildly injured viable but nonculturable cells.

J Dairy Sci, 1997 Oct, 80(10), 2398 - 402
Responses of antibody titers to intramammary immunization with Escherichia coli J5 bacterin; Hogan JS et al.; The effect of an immunization schedule on responses of antibody titers was tested following vaccination with an Escherichia coli J5 bacterin . Eighteen cows were equally distributed among three immunization schedules: 1) subcutaneous injection at 14 d prior to the end of lactation, intramammary immunization at 7 d after drying off, and subcutaneous injection at 30 d into the dry period; 2) subcutaneous injections at drying off, at 30 d into the dry period, and within 12 h after calving; and 3) unimmunized controls . The E . coli J5 bacterin consisted of 5 ml of 10(9) boiled cells/ml of 0.9% NaCl plus 0.005% phenol emulsified with 5 ml of Freund's incomplete adjuvant . Subcutaneous injections were administered on the upper part of the rib cage, posterior to the scapula . Intramammary immunizations of 2.5 ml of bacterin were infused via the teat canal into each of the four mammary glands . Intramammary immunization increased rectal temperatures at 12 h after infusion, but subcutaneous injections did not induce febrile responses . Intramammary immunization enhanced immunoglobulin G titers in serum and whey on d 0 of lactation compared with subcutaneous immunizations . Immunoglobulin G titers in serum also were greater at d 30 of the dry period and at d 14 and 21 of lactation for cows that received intramammary immunization than for cows that were vaccinated by subcutaneous injections only . Immunoglobulin M titers in whey and serum on d 21 of lactation were greater for cows that received intramammary immunizations than for cows that were immunized by subcutaneous injections only.

Diagn Mol Pathol, 1997 Aug, 6(4), 199 - 208
Bcl-2 expression and DNA fragmentation in breast carcinoma, pathologic and steroid hormone receptors correlates; Diaz-Cano SJ et al.; B-cell leukemia/lymphoma (bcl-2) expression can override the apoptosis development in lymphoid and hormonally regulated tissue-like breast . The presence of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) have revealed in breast carcinomas, but they have not been correlated to the bcl-2 protein expression and DNA fragmentation markers . We evaluated the immunohistochemical expression of bcl-2 protein and hormonal receptors (ER, PR, AR) and differentiation grade in 37 infiltrating ductal carcinomas of the breast for which frozen tissues were available for DNA extraction . The immunohistochemical reaction for bcl-2 was considered positive if more than 50% of neoplastic cells had intense cytoplasmic staining, whereas for steroid receptor evaluation Battifora's criteria were used . The DNA was extracted according to the phenol-chloroform procedure and used for bcl-2 gene rearrangement study of the major breakpoint region (Southern blot) and for membrane-based end-labeling using digoxigenin-labeled nucleotides and E . coli DNA polymerase I (Klenow fragment) . The results were quantified by three different observers . Low-grade carcinomas were positive for bcl-2 protein (27/28, 96.4%) and ER (15/28, 53.6%), whereas the remaining neoplasms were negative for bcl-2 (9/9, 100.0%) and ER (8/9, 53.6%) (p < 0.001) . No statistically significant differences were revealed at the bcl-2, PR and AR comparisons . The Southern blot analysis for bcl-2 major breakpoint region showed neither rearrangement nor genetic amplification (densitometric study) . Only the membrane-based end-labeling of DNA fragments showed correlation with bcl-2 protein and ER expressions: all except one bcl-2-negative tumor and two bcl-2-positive tumors had positive labeling using 7 pg of DNA at dot blot analysis (p < 0.002) . The bcl-2 protein expression would allow both proliferation and cell progression by blocking apoptosis in well-differentiated, ER-positive breast carcinomas . In these neoplasms, DNA fragmentation as a molecular marker of apoptosis was prevented by bcl-2 expression.

Anim Reprod Sci, 1997 Jul, 47(4), 337 - 42
The potential transmission of infectious agents by semen packaging during storage for artificial insemination; Russell PH et al.; Plastic straws, of a type widely used for semen cryopreservation, sealed using three different methods, (PVA powder, plastic spheres and plasticine modelling clay) were tested for leakage of low molecular weight dye (methylene blue), bacteria (Escherichia coli) and virus (Newcastle disease virus) . Leakage was found to be dependent on the method used to fill the straws . Straws filled using a traditional 'dip and wipe' method and sealed with PVA powder demonstrated a significant degree of methylene blue leakage (0.0269% of the total straw contents) probably associated with contamination of the powder sealing plug . Straws filled using an aseptic filling technique showed no detectable leakage of any agent with any of the sealing methods . This study highlights the need to establish good-practice guidelines for the packaging of semen collected for freezing and future AI from non-domestic livestock where disease-free status cannot be guaranteed and unsophisticated technology is used.

Acta Biochim Pol, 1997, 44(2), 309 - 14
Studies on the nature of genotoxic and cytotoxic effects induced by hydralazine; Marczewska J et al.; The nature of genotoxic and cytotoxic effects induced by hydralazine was analyzed taking into account possible protection of cells by catalase, superoxide dismutase and dimethyl sulfoxide . For the experiments designed to evaluate the influence of scavengers on the genotoxicity expressed as the SOS induction factor the E . coli PQ37 strain was used . The cytotoxic effects were investigated in V3 cells cultured in vitro . The genotoxicity and cytotoxicity of hydralazine were suppressed by catalase in a dose-dependent manner but they were enhanced by superoxide dismutase . No protective effect of dimethyl sulfoxide was observed . Our results indicate that H2O2 plays an essential role in the genotoxicity and cytotoxicity of hydralazine.

Acta Biochim Pol, 1997, 44(2), 275 - 83
Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants; Bolewska K et al.; Calcium binding S100A1 protein consists of two S100 alpha subunits . On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain . In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues . A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid . The gene was expressed in Escherichia coli utilizing the T7 expression system . The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini . Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine . The material was partly oxidized . Interestingly, only the homodimers of a, b, and c species were formed . The total yield of the protein was about 50 mg/l of culture . Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system . In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained . The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half . As expected, the mutants of S100 alpha subunits bind only one calcium ion.

Acta Biochim Pol, 1997, 44(2), 221 - 9
Expression, purification and kinetic properties of human recombinant phospholipase C delta 3; Pawelczyk T et al.; To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E . coli cells as a (6 x His) fusion protein and purified to homogeneity . From 1 L of E . coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography . The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3 . The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively . Recombinant PLC delta 3 showed an absolute requirement for Ca2+ . Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity . In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration . The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively . The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin . PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1 . The data presented indicate that the expression of PLC delta 3 in E . coli cells permits to obtain active enzyme . The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.

Mutat Res, 1997 Sep, 382(1-2), 35 - 43
Search for DNA sequence variations using a MutS-based technology; Bellanne-Chantelot C et al.; The search for DNA sequence variations (DSV) is emphasized with genetic studies of a large number of multifactorial diseases . Saturation of regions of interest with diallelic polymorphisms will be an essential step to pinpoint, through association studies, predisposing genes . We have developed a solid-phase method based on the ability of mismatch binding protein MutS to recognize single nucleotide mismatches . This approach was applied to the study of 83 sequence-tagged sites (STSs) extracted from an eight centimorgans (cM) chromosome 21 region . One-third of tested STSs were found to be polymorphic leading to a frequency of one DSV every 822 base pairs (bp) . Sequencing of analyzed STSs showed the high reliability of the MutS-based technology for mismatches up to 2 bp in DNA fragments ranging in size from 200 bp to 1 kilobase (kb) . The entire assay which is performed in a solid-phase format without the need of electrophoresis or sequencing, will provide an efficient tool for new polymorphism detection.

Mutat Res, 1997 Sep, 382(1-2), 21 - 33
The design of a new mutation model for active genes: expression of the Escherichia coli lac operon in mammalian cells; van Sloun PP et al.; The design of a novel transgenic mouse model is described that should allow analysis of mutations at a single cell level in all tissues of a model animal . The model is based on the correct regulation of the Escherichia coli lac operon in mammalian cells . Induction of a mutation in the lacI gene will result in the loss of transcriptional repression of the lacZ gene in mutated cells . Expression of beta-galactosidase can subsequently be detected at the single cell level . The model was first tested in vitro using transfection of mouse LTK- cells . LacZ expression was very heterogeneous in most of the stable transfectants and seemed to be subject to epigenetic inactivation . One clone (IIB1) was isolated that stably expressed lacZ in more than 99% of its cells . Subsequent introduction of the lacI gene into IIB1 cells resulted in correct transcriptional repression of the lacZ gene that could be alleviated by IPTG, an allosteric inducer of lacI repression . However, in time the extent of beta-galactosidase induction gradually declined suggesting that the prolonged repressed transcriptional state triggers epigenetic inactivation . Variegated expression of the lacZ gene was not confined to cultured cells since several transgenic lines also did not express the lacZ transgene . This study shows that while the susceptibility of the lacZ gene to inactivation processes poses a fundamental problem, correct regulation of the expression of a reporter gene by the lacI repressor protein is feasible in mammalian cells when assayed at the single cell level . Thus, the model can in principle be used for the detection of mutagenic events at the lacI locus . Targeting of the lacZ gene to an endogenous housekeeping gene might prevent epigenetic inactivation . Alternatively, with the use of another reporter gene in the mutation detection system the proposed transgenic mouse model could be realized.

Nat Struct Biol, 1997 Nov, 4(11), 887 - 91
Crystal structure of DNA photolyase from Anacystis nidulans; Tamada T et al.; The crystal structure at 1.8 A resolution of 8-HDF type photolyase from A . nidulans shows a backbone structure similar to that of MTHF type E . coli photolyase but reveals a completely different binding site for the light-harvesting cofactor.

J Diarrhoeal Dis Res, 1997 Jun, 15(2), 53 - 8
Human colostrum IgA antibodies reacting to enteropathogenic Escherichia coli antigens and their persistence in the faeces of a breastfed infant; Carbonare SB et al.; IgA antibodies reacting to enteropathogenic Escherichia coli (EPEC) antigens in human colostrum and their role in the inhibition of EPEC adherence to HEp-2 cells were studied . Colostrum IgA was isolated with a Sepharose anti-IgA column . IgA-depleted colostrum lost its inhibitory effect on EPEC adhesion, while the IgA-enriched eluate was a potent adherence inhibitor . The same eluate showed a significant loss of inhibitory activity after absorption with an EPEC strain showing localised adherence (LA+), but no alteration after absorption with an LA- strain . No bands were observed in Western blot analysis with LA+ absorbed eluate and with a crude extract of the EPEC strain, but the eluate absorbed with LA- showed a strong recognition of a 94-kDa band, a molecular weight equivalent to that of intimin . Colostrum antibodies reacting to non-protein antigens were not detected by Western blot analysis . The persistence of anti-EPEC IgA in the gastrointestinal tract was shown by the strong reactivity to the 94-kDa band in Western blot analysis of one mother's colostrum and her infant's faeces . These data confirm the role of colostrum antibodies in protecting the neonate against infections due to EPEC.

Biochemistry (Mosc), 1997 Aug, 62(8), 890 - 7
Expression of cDNA fragment encoding ligand-binding domain of human low density lipoprotein receptor in Escherichia coli cells; Runova OL et al.; To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains . To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli . We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically . Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).

Biochem J, 1997 Oct 15, 327 ( Pt 2), 593 - 600
Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid; Liu LF et al.; Escherichia coli-expressed chicken-liver glutathione S-transferase, cGSTA1-1, displays high ethacrynic acid (EA)-conjugating activity . Molecular modelling of cGSTA1-1 with EA in the substrate binding site reveals that the side chain of Phe-111 protrudes into the substrate binding site and possibly interacts with EA . Replacement of Phe-111 with alanine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in EA-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (DeltaDeltaG) of 4.0 kJ/mol lower than that of the wild-type cGSTA1-1 . Two other amino acid residues that possibly interact with EA are Ser-208 and Lys-15 . Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjugating activity (2.0 mmol/min per mg) and an incremental Gibbs free energy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant . The cGSTA1-1(F111A) mutant, with an additional Lys-15-to-leucine substitution, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg) . The Km values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants for EA are nearly identical . The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min per mg) . The kcat of this reaction can be increased 2 . 5-fold by substituting Arg-15 and Glu-104 with lysine and glycine respectively . The KmEA of the cGSTA2-2(R15KE104G) double mutant is nearly identical with that of the wild-type enzyme . Another double mutant, cGSTA2-2(E104GL208S), has a KmEA that is 3.3-fold lower and a kcat that is 1.8-fold higher than that of the wild-type enzyme . These results, taken together, illustrate the interactions of Lys-15 and Ser-208 on cGSTA1-1 with EA.

Biochem J, 1997 Oct 15, 327 ( Pt 2), 407 - 12
Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine; Ogawa H et al.; An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli . Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture . HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking . The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation {Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) Eur . J . Biochem . 168, 141-151} . In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested {Ogawa and Fujioka (1982) J . Biol . Chem . 257, 3447-3452}, the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH . The Hill coefficient increased with increasing pH and a pKa of 8.11 was obtained in this transition . The values of Vmax and Km for glycine were not different between the two enzymes . These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour.

Perit Dial Int, 1997 Sep-Oct, 17(5), 455 - 66
Absence of modulating effects of cytokines on antioxidant enzymes in peritoneal mesothelial cells; Chen JY et al.; OBJECTIVE: To investigate the modulation of superoxide dismutase, glutathione peroxidase, and catalase by cytokines and endotoxin in human peritoneal mesothelial cells . DESIGN: Cultured human peritoneal mesothelial cells were treated with various concentrations of interleukin-1 alpha, tumor necrosis factor-alpha (TNF-alpha), interleukin-6, interleukin-8, transforming growth factor-beta (TGF beta), and lipopolysaccharide . Cell morphology was observed and the activities of superoxide dismutase, catalase, and glutathione peroxidase were assayed . The antioxidant enzyme activities of human peritoneal mesothelial cells were also compared with those of human liver and kidney tissues . RESULTS: Interleukin-1 alpha, TNF alpha, TGF beta, and lipopolysacharide caused dose-dependent cytotoxicities in mesothelial cells . The activities of these three antioxidant enzymes did not change after treatment with cytokines and endotoxin . The total superoxide dismutase activity of confluent human peritoneal mesothelial cells was found to be greater than that of human liver and kidney tissues and was composed mostly of manganese superoxide dismutase activity . Furthermore, glutathione peroxidase and catalase activities of human peritoneal mesothelial cells were lower than those of human liver and kidney tissues . CONCLUSION: In human peritoneal mesothelial cells, lack of induction of antioxidant enzymes by inflammatory cytokines, as well as high superoxide dismutase activity accompanied by insufficient glutathione peroxidase and catalase activities may both contribute to the susceptibility of these cells to oxidative damage . Therefore, appropriate management to decrease oxidative injury to the peritoneum should be taken into consideration when treating long-term continuous ambulatory peritoneal dialysis patients.

J Immunoassay, 1997 Nov, 18(4), 321 - 33
A simple and rapid immunoassay system using green fluorescent protein tag; Aoki T et al.; A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli . The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions . However, heat-denatured GFP-NSE was non-fluorescent and migrated at 74 kDa corresponding to the theoretical value . This suggests that the special structure of GFP, which is not denatured by SDS, influences its mobility in SDS-PAGE under non-heating conditions . The fluorescence intensity of GFP-NSE was measurable over a wide range by spectrophotometry or densitometry . The competitive immunoassay for NSE was performed using GFP-NSE as labeled antigen . Under our assay conditions, the working range of this system was about 2 -60 ng . This simple and rapid fluorescence immunoassay (FIA) using GFP-tagged antigen may be applicable to many protein markers.

Immunotechnology, 1997 Oct, 3(3), 217 - 26
Regulation and expression of human Fabs under the control of the Escherichia coli arabinose promoter, PBAD; Clark MA et al.; BACKGROUND: The L-arabinose operon from E . coli contains an inducible promoter PBAD which has been extensively studied for the control of gene expression . PBAD has a number of potential advantages over Plac, and has been used successfully to promote high level expression of recombinant proteins . OBJECTIVES: The aim of this study was to investigate PBAD as an alternative system to Plac for the bacterial expression of recombinant Fabs . STUDY DESIGN: The promoter PBAD from the E . coli arabinose operon araBAD and the gene encoding the regulator of this promoter, were cloned into the phagemid expression vector MCO1 . Expression of human recombinant tetanus toxoid (TT) and c-erbB2 Fabs under the control of PBAD was compared at two induction temperatures with the same Fabs produced under the control of Plac . RESULTS: Expression of TT and c-erbB2 Fabs under the control of PBAD was comparable to Fab expression from Plac . However, highly expressed TT Fab under the control of PBAD was localised to the soluble periplasmic fraction whereas under the control of Plac, there was greater leakage of Fab into the culture supernatant . In addition, Fab expression from PBAD could be more tightly repressed than from Plac . CONCLUSION: PBAD is a useful and cheaply inducible alternative to the more commonly used Plac for the rapid expression of soluble recombinant human antibody fragments.

Immunotechnology, 1997 Oct, 3(3), 173 - 84
A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells; Brocks B et al.; Single chain antibodies (scFv) are usually produced in E . coli, but generation of certain scFv derivatives, such as complex fusion proteins or glycosylated forms of scFv is restricted to eukaryotic expression systems . We investigated the production of soluble mono- and bivalent single chain antibodies (scFv) in eukaryotic cells and describe a cassette vector system for mammalian and baculovirus expression which is compatible with an established vector system for bacterial expression and phage display selection of scFvs . The applied model scFv was derived from a murine antibody (H398) against human tumor necrosis factor receptor 1 (TNFR60), known to be a potent antagonist of TNF action in its monomeric form and a potential therapeutic agent for treatment of TNF-mediated diseases . Surprisingly, the monomeric scFv form of H398 (scFv H398) is expressed but not secreted in different mammalian cells . In contrast, in insect cells using recombinant baculovirus, a monovalent scFv H398 and a bivalent scFv fusion protein with an human IgG1 Fc region were expressed and secreted with correctly processed signal sequence . Concerning the influence of valency of the model Ab and its derivatives on antigen binding affinity and neutralisation of TNF activity, we found that the mono- and bivalent form of scFv H398 possesses the same characteristics as proteolytically produced Fab H398 and original mAb H398, respectively . Furthermore, fusion of the Ig Fc protein to scFv H398 increase the in vitro half-life at 37 degrees C . We conclude that the described cassette vectors readily allow the eukaryotic expression of mono- and bivalent scFv derivatives to analyse the influence of valency of scFv molecules on antigen binding and biological activity.

Eur J Drug Metab Pharmacokinet, 1997 Jul-Sep, 22(3), 223 - 7
The hepatic and duodenal activities of some drug metabolizing enzymes in chickens: influence of infection with Escherichia coli endotoxin and coccidiosis; Ali BH; The activities of the drug metabolizing enzymes aminopyrine-N-demethylase, aniline hydroxylase and UDP-glucuronyl transferase, together with protein concentration, were measured in liver microsomes and duodenal mucosa from healthy chickens and chickens experimentally infected with Escherichia coli endotoxin, or naturally infected with Eimeria necatrix and Eimeria tenella (clinically classified as slight, moderate or severe infections) . E . coli (2 mg/kg, 3 days) and severe coccidiosis significantly decreased the activities of the three enzymes in the liver and duodenum . However, infection by the E . coli endotoxin at lower doses (0.5 and 1 mg/kg, 3 days) and moderate or slight coccidial infections had no significant effect on the activities of these enzymes . Neither E . coli nor coccidial infections significantly affected the liver/body weight ratio . However, infection with the E . coli endotoxin (2 mg/kg, 3 days) and the moderate or severe coccidial infections significantly reduced microsomal protein concentration in the liver.

J Lab Clin Med, 1997 Oct, 130(4), 442 - 9
Helicobacter pylori induces proinflammatory cytokines and major histocompatibility complex class II antigen in mouse gastric epithelial cells; Maekawa T et al.; Although Helicobacter pylori has been reported to stimulate the release of various cytokines from gastric tissue, it remains unknown whether normal and nontumorous gastric epithelial cells produce these cytokines . Therefore, in this study, we used a normal mouse gastric surface mucous cell line (GSM06) to determine whether gastric epithelial cells produce proinflammatory cytokines in response to H . pylori . The expression of MHC class II antigen was also examined, to investigate whether gastric epithelial cells participate in the immune response to H . pylori . In the study, GSM06 cells were incubated with H . pylori or its lipopolysaccharide (LPS) . Proinflammatory cytokines were detected by Northern and Western blot analysis . The expression of MHC class II antigen was examined by fluorescence activated cell sorter (FACS) analysis . Genetic expression of proinflammatory cytokines such as interleukin-1alpha, tumor necrosis factor-alpha, and cytokine-induced neutrophil chemoattractant-2beta was enhanced by both intact and sonicated H . pylori, but not by H . pylori LPS . The expression of MHC class II antigen was induced by H . pylori more strongly than by interferon-gamma . We conclude that H . pylori induces the expression of proinflammatory cytokines and MHC class II antigen in gastric epithelial cells . Gastric epithelial cells may act as antigen-presenting cells and participate in the immune response to H . pylori infection.

Gene, 1997 Oct 15, 199(1-2), 203 - 10
Characterization of the gntT gene encoding a high-affinity gluconate permease in Escherichia coli; Izu H et al.; We characterized the gntT gene encoding a high-affinity gluconate permease of Escherichia coli K-12 . Primer extension and lacZ-operon fusion analyses revealed that gntT has one strong and two weak promoters, all of which are regulated positively by cAMP-CRP and negatively by GntR . The weak promoters became constitutive when separated from the upstream region including the strong promoter that overlaps a putative GntR-binding sequence . Gluconate-specific uptake activity was observed with cells harboring the gntT plasmid clone, which was enhanced by the presence of gntK encoding gluconate kinase.

Gene, 1997 Oct 15, 199(1-2), 83 - 91
The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein; Thomas PW et al.; A heat shock protein-encoding gene (hsp60) from the human respiratory fungal pathogen, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells . Both the genomic and cDNA sequences are presented . The transcription start point and poly (A) addition site were confirmed . The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and pI of 5.6 . The translated protein revealed two potential N-glycosylation sites . The deduced HSP60 showed 78-83% aa sequence similarity to reported fungal HSP60 proteins . The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization . Expression of a 1737-bp cDNA fragment of the hsp60 gene in E . coli resulted in production of a recombinant protein . Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene . Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci . The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals . The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.

Gene, 1997 Oct 15, 199(1-2), 19 - 23
Identification of a novel Escherichia coli gene whose expression is dependent on the flagellum-specific sigma factor, FliA, but dispensable for motility development; Ide N et al.; FliA is an alternative sigma factor specific for class 3 flagellar operons . Using a promoter-probe vector, we randomly cloned Escherichia coli DNA fragments, which showed FliA-dependent promoter activities . Among the DNA fragments cloned, one was found to be derived from a non-flagellar region . Hybridization analysis with the Kohara E . coli library indicated that this DNA fragment is located at around 35.4 min on the E . coli chromosome where no flagellar gene has been reported yet . DNA sequence analysis revealed that it contains an FliA-dependent promoter-like sequence followed by an open reading frame (ORF) that can encode a 110-amino-acid protein . A rho-independent terminator-like sequence follows this ORF . This putative gene was named flxA . A gene disruptant was constructed by inserting the kan gene cassette into the flxA gene on the chromosome . This mutant was found to be actively motile, suggesting that this gene is unlikely to be involved in the motility phenotype of E . coli.

FEBS Lett, 1997 Oct 6, 415(3), 317 - 20
Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli; Eppens EF et al.; Porins form voltage-gated channels in the bacterial outer membrane . These proteins are composed of three identical subunits, each forming a 16-stranded beta-barrel . In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied . The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined . Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.

FEBS Lett, 1997 Oct 6, 415(3), 289 - 93
Affinity and folding properties both influence the selection of antibodies with the selectively infective phage (SIP) methodology; Pedrazzi G et al.; We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology . As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines . The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity . After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected . SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.

Biochim Biophys Acta, 1997 Sep 5, 1341(2), 165 - 72
Aspartate 155 of human transketolase is essential for thiamine diphosphate-magnesium binding, and cofactor binding is required for dimer formation; Wang JJ et al.; Active human transketolase is a homodimeric enzyme possessing two active sites, each with a non-covalently bound thiamine diphosphate and magnesium . Both subunits contribute residues at each site which are involved in cofactor binding and in catalysis . His-tagged transketolase, produced in E . coli, was similar to transketolase purified from human tissues with respect to Km apps for cofactor and substrates and with respect to cofactor-dependent hysteresis . Mutation of aspartate 155, corresponding to a conserved aspartate residue among thiamine diphosphate-binding proteins, resulted in an inactive protein which could not bind the cofactor-magnesium complex and which could not dimerize . The results are consistent with the suggestion that aspartate 155 is an important coordination site for magnesium . In support of this interpretation, binding of cofactor by wild type apo-transketolase required the presence of magnesium . Additionally, monomeric apo-his-transketolase required both magnesium and cofactor binding for dimer formation.

Am J Physiol, 1997 Oct, 273(4 Pt 1), L889 - 94
rhs-TM prevents ET-induced increase in pulmonary vascular permeability through protein C activation; Uchiba M et al.; We have previously demonstrated that recombinant human soluble (rhs) thrombomodulin (TM) inhibits the endotoxin (ET)-induced increase in pulmonary vascular permeability by inhibiting leukocyte activation . In the present study, we examined whether rhs-TM could inhibit the ET-induced increase in pulmonary vascular permeability in rats by activating protein C . rhs-TM did not inhibit ET-induced increases in pulmonary vascular permeability when its protein C activation ability was selectively inhibited by a monoclonal antibody (MAb) against rhs-TM (MAb R5G12) . Histological examination revealed that neutrophil infiltration in lung tissues after ET administration was significantly reduced by rhs-TM, but infiltration was not reduced by MAb R5G12-pretreated rhs-TM . ET-induced intravascular coagulation was prevented by rhs-TM and by MAb R5G12-pretreated rhs-TM . However, ET-induced coagulation was not prevented by rhs-TM that had been treated with MAb F2H5, which cannot bind thrombin or activate protein C . These observations strongly suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes through thrombin binding and the subsequent protein C activation and may prevent ET-induced intravascular coagulation through thrombin binding.

Am J Physiol, 1997 Oct, 273(4 Pt 1), L807 - 13
Regulation of cGMP by soluble and particulate guanylyl cyclases in cultured human airway smooth muscle; Hamad AM et al.; Although guanosine 3',5'-cyclic monophosphate (cGMP) acts as a relaxant second messenger, the regulation of intracellular cGMP has not been comprehensively studied in human airway smooth muscle . We studied the production of cGMP by cultured human airway smooth muscle cells (HASMC) after stimulation with activators of soluble guanylyl cyclase {sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP)} and particulate guanylyl cyclase {atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Escherichia coli heat stable enterotoxin (STa)} . cGMP was measured by enzyme-linked immunosorbent assay . Both SNP (10(-6) to 10(-3) M) and SNAP (10(-6) to 10(-3) M) caused concentration-dependent elevation of cGMP in the presence of the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (10(-3) M), with cGMP increasing 6- and 15-fold in response to SNP and SNAP, respectively, at the highest concentration tested (10(-3) M) . The increases in cGMP in response to SNP (5 x 10(-5) M) and SNAP (10(-5) M) were inhibited by hemoglobin (Hb; 5 x 10(-5) M), a nitric oxide scavenger, and methylene blue (MB; 5 x 10(-4) M), an inhibitor of guanylyl cyclase . cGMP accumulation after SNAP was abolished by both Hb and MB . The response to SNP was inhibited by 79% with Hb and was abolished with MB . ANP, BNP, and CNP (10(-9) to 10(-5) M) + phosphoramidon (10(-6) M) caused a concentration-dependent elevation in cGMP with an order of potency ANP > BNP > CNP . cGMP formation in the presence of the highest concentration of the most potent natriuretic peptide (10(-5) M ANP) was two- to threefold greater than with the highest concentration of SNAP . The increase in cGMP seen with natriuretic peptides was similar in the presence or absence of phosphoramidon, a neutral endopeptidase (NEP) inhibitor, suggesting that NEP is not playing a role in modulating the effect of natriuretic peptides in HASMC . STa (400 IU/ml) had no effect on cGMP levels . SNAP- and ANP-induced cGMP accumulation was increased by the selective type V PDE inhibitors SKF-96231 and zaprinast, suggesting that type V PDE is responsible for cGMP breakdown in HASMC . These results suggest that cultured HASMC contain both soluble and particulate guanylyl cyclases . The order of potency of the natriuretic peptides ANP > BNP > CNP suggests that type A particulate membrane-bound guanylate cyclase predominates.

Am J Physiol, 1997 Oct, 273(4 Pt 1), C1160 - 7
Activation of NF-kappaB in intestinal epithelial cells by enteropathogenic Escherichia coli; Savkovic SD et al.; The initial response to infection is recruitment of acute inflammatory cells to the involved site . Interleukin (IL)-8 is the prototypical effector molecule for this process . Transcription of the IL-8 gene is primarily governed by the nuclear transcription factor (NF)-kappaB . Intestinal epithelial cells produce IL-8 in response to infection by enteric pathogens yet remain quiescent in a milieu where they are literally bathed in normal bacterial flora . We therefore sought to investigate NF-kappaB activation in response to enteropathogenic Escherichia coli (EPEC), nonpathogenic E . coli, and bacterial lipopolysaccharide in an intestinal epithelial cell (T84) model and to determine whether EPEC-induced activation of NF-kappaB factor is causally linked to IL-8 production . We report herein that NF-kappaB is activated by EPEC, yet such a response is not extended to nonpathogenic organisms or purified E . coli lipopolysaccharide . Transcription factor decoys significantly diminished IL-8 production in response to EPEC, demonstrating a causal relationship . Furthermore, deletion of specific EPEC virulence genes abrogates the NF-kappaB-activating property of this pathogen, suggesting that specific bacterial factors are crucial for inducing this response . These studies show for the first time that infection of intestinal epithelial cells with EPEC activates NF-kappaB, which in turn initiates IL-8 transcription, and highlight the differential response of these cells to bacterial pathogens vs . nonpathogens.

Biotechnol Appl Biochem, 1997 Oct, 26 ( Pt 2), 117 - 23
Purification of mouse H1 histones expressed in Escherichia coli; Wellman SE et al.; We amplified the coding regions of the previously cloned H1 genes H1-1, H1 zero and H1t and inserted them into the expression vector pET-11d . The synthesis of the H1 histones can be induced in the appropriate strains of bacteria, and the H1 histones can be readily purified . We report detailed protocols for the purification of the expressed proteins using combinations of ion-exchange and reverse-phase HPLC . Sufficient amounts of each pure variant protein can be obtained for use in physical studies of H1-DNA interactions.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12497 - 502
The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli; Bates DB et al.; Despite the widely accepted view that transcription of gid and mioC is required for efficient initiation of cloned oriC, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal oriC . Furthermore, neither gid nor mioC transcription is required in cells deficient in the histone-like proteins Fis or IHF . However, oriC that is sufficiently impaired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes . We conclude that transcription of mioC and especially gid is needed to activate oriC only under suboptimal conditions . We suggest that either the rifampicin-sensitive step of initiation is some other transcription occurring from promoter(s) within oriC, or the original inference of transcriptional activation derived from the rifampicin experiments is incorrect.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12485 - 90
Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1; Chen F et al.; The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis . We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase . Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP . This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro . Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted . Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe . Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner . Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage . Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12348 - 53
Novel mechanism for carbamoyl-phosphate synthetase: a nucleotide switch for functionally equivalent domains; Kothe M et al.; Carbamoyl-phosphate synthetases (CPSs) utilize two molecules of ATP at two internally duplicated domains, B and C . Domains B and C have recently been shown to be structurally {Thoden, J . B., Holden, H . M., Wesenberg, G., Raushel, F . M . & Rayment, I . (1997) Biochemistry 36, 6305-6316} and functionally {Guy, H . I . & Evans, D . R . (1996) J . Biol . Chem . 271, 13762-13769} equivalent . We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A/B motif in domains B and C . To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS . In this mechanism utilization of ATP bound to domain C is coupled to carbamoyl-phosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.

Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12280 - 4
The ribosome-in-pieces: binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA; Munishkin A et al.; An oligoribonucleotide (a 27-mer) that mimics the sarcin/ricin (S/R) domain of Escherichia coli 23S rRNA binds elongation factor EF-G; the Kd is 6.9 microM, whereas for binding to ribosomes it is 0.7 microM . Binding saturates when EF-G and the S/R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G . Binding of EF-G to S/R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP . The effects of mutations of the S/R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides . EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S/R and thiostrepton oligoribonucleotides.

Virology, 1997 Oct 27, 237(2), 327 - 36
RNA binding activity of NIa proteinase of tobacco etch potyvirus; Daros JA et al.; The C-terminal domain of NIa protein (NIaPro) from tobacco etch potyvirus (TEV) is a sequence-specific proteinase required for processing of the viral polyprotein . This proteinase also interacts with NIb, the TEV RNA-dependent RNA polymerase . NIaPro and two NIaPro-containing polyproteins (NIa and 6/NIa) were analyzed from extracts of recombinant Escherichia coli . Using RNA-protein blot and UV-crosslinking assays, NIaPro and the NIaPro-containing polyproteins were shown to possess RNA-binding activity . NIaPro bound nonspecifically to several RNAs, including plus- and minus-strands of the TEV 5' and 3' noncoding regions . Saturation binding data obtained using the UV-crosslinking assay were consistent with a possible cooperative RNA-binding activity of NIaPro . In addition, the RNA-binding activities of NIaPro and full-length NIa protein were similar . Based on its RNA-binding activity and other known functions, NIaPro or a NIaPro-containing polyprotein is proposed to serve one or more direct roles during TEV RNA synthesis .

Virology, 1997 Oct 27, 237(2), 283 - 95
Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins; Rojas MR et al.; The role of bean common mosaic necrosis potyvirus (BCMNV) and lettuce mosaic potyvirus (LMV) proteins was investigated in terms of their capacity to function as viral movement proteins (MPs) . Using Escherichia coli-expressed proteins and microinjection techniques, direct evidence was obtained that both the potyviral capsid protein (CP) and helper component- proteinase (HC-Pro) function in this capacity, in that both proteins (a) trafficked from cell to cell, (b) induced an increase in plasmodesmal size exclusion limit, and (c) facilitated cell-to-cell movement of viral RNA . CP and HC-Pro mutants were also produced and used in microinjection experiments . Mutations in the core region of the CP either impaired (single and double amino acid substitution mutants) or abolished (triple amino acid substitution mutant) cell-to-cell movement, as did C-terminal deletion mutants in HC-Pro . The BCMNV P1, CI, NIa, and NIb proteins did not exhibit viral MP properties, but NIa and NIb proteins were found to accumulate within the nuclei of injected cells . These results further establish the multifunctional nature of the potyvirus CP and HC-Pro .

J Struct Biol, 1997 Oct, 120(1), 105 - 8
Crystallization and preliminary X-ray analysis of human D-dopachrome tautomerase; Sugimoto H et al.; D-Dopachrome tautomerase catalyzes the conversion of D-dopachrome to 5,6-dihydroxyindole . This protein has amino acid sequence homology with that of macrophage migration inhibitory factor (MIF), suggesting a pathophysiological role of this protein in inflammatory and immunological events . We previously determined the tertiary structure of MIF and revealed the functional and evolutional relationships of this protein to isomerase . However, the reaction mechanism of both proteins associated with the inflammatory response, immune system, or tautomerase activities in vitro have not yet been clarified . The tertiary structure of D-dopachrome tautomerase would provide insight into the molecular function and the mechanism of these proteins . In this study, we crystallized human D-dopachrome tautomerase by a hanging-drop vapor diffusion method . The crystals belong to the trigonal space group P3, with unit cell dimensions a = b = 84.2 A and c = 41.0 A . They contain three (or two) monomers in the asymmetric unit, corresponding to a VM value of 2.21 (or 3.32) A3 Da-1 . The best crystals diffract X-ray to 1.6 A resolution using a synchrotron radiation source . Crystallization of the selenomethionyl derivative of the protein for applying the multiwavelength anomalous diffraction method was also successful.

J Magn Reson, 1997 Oct, 128(2), 101 - 4
Dynamic frequency shifts of complexed ligands: An NMR study of D--1-13C,1-2H-glucose complexed to the Escherichia coli periplasmic glucose/galactose receptor; Gabel SA et al.; The 13C multiplet structure of D--1-13C,1-2H-glucose complexed to the Escherichia coli periplasmic glucose/galactose receptor has been studied as a function of temperature . Asymmetric multiplet patterns observed are shown to arise from dynamic frequency shifts . Multiplet asymmetry contributions resulting from shift anisotropy-dipolar cross correlations were found to be small, with optimal fits of the data corresponding to small, negative values of the correlation factor, chiCD-CSA . Additional broadening at higher temperatures most probably results from ligand exchange between free and complexed states . Effects of internal motion are also considered theoretically, and indicate that the order parameter for the bound glucose is >/=0.9 .

J Mol Biol, 1997 Oct 31, 273(3), 572 - 85
Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides; Reedstrom RJ et al.; Fluorescence-based solution methods have been used to study the binding of the trp repressor of Escherichia coli to a series of oligonucleotides bearing all or partial determinants for high affinity specific binding . The tryptophan, salt concentration and competitor DNA dependence of the binding affinities was examined for these targets . Binding to a fluorescein-labeled 20 base-pair hairpin structure oligonucleotide, which contains a palindromic repressor binding site (GAACTAGTTAACTAGTAC) and is known to bind repressor in a 1 : 1 dimer-DNA complex, resulted in a protein concentration-dependent, competable static quenching of fluorescence in presence of co-repressor, l-tryptophan . The affinity recovered from the fits of these intensity profiles at 100 mM KCl was on the order of 4x10(8) M-1 . In absence of co-repressor an increase in intensity at high repressor concentration (>10(-7) M) was observed . The salt concentration dependence of the specific binding of the holo-repressor to this oligonucleotide was approximately half as large as what would be predicted by the number of phosphate contacts in the crystal structures of the complex . Repressor binding to the fluorescein-labeled hairpin 20mer was compared with binding to a rhodamine-labeled 36 base-pair oligonucleotide bearing two inverted structural half-sites GNACT separated by an eight base-pair spacer containing none of the natural intervening sequence . The rather low affinity observed for the 36mer revealed that the intervening sequence in the natural operators contains energetic specificity determinants . Binding to a rhodamine-labeled oligonucleotide bearing a completely non-specific sequence was shown to occur over the same concentration range (>100 nM), regardless of tryptophan concentration, whereas binding to sequences bearing partial specificity ratio between 100 and 1000, depending upon the salt concentration . Even in absence of added KCl, the specificity ratio of trp repressor was greater than 100, implicating a significant free energy contribution from non-electrostatic interaction forces .

Anal Biochem, 1997 Nov 1, 253(1), 103 - 11
Nonspecific amine immobilization of ligand can Be a potential source of error in BIAcore binding experiments and may reduce binding affinities; Kortt AA et al.; The interaction of monovalent forms of NC41, an anti-viral neuraminidase antibody, and the antiidiotype antibody 11-1G10 has been used as a model system for BIAcore analysis to demonstrate the potential problems resulting from the nonspecific amine coupling procedure . To avoid complications due to antibody bivalency, monovalent Fab fragments and monomeric recombinant scFvs were used . When immobilized by amine coupling, the 11-1G10 anti-idiotype fragments were found to have an artificially reduced affinity for NC41 compared to the results obtained using site-directed immobilization via C-terminal thiol residue and from solution equilibrium measurements . The NC41 antibody fragments, on the other hand, were able to retain their 11-1G10 binding affinity when immobilized nonspecifically through free amine groups . These data, in combination with the known sequences of the two antibodies, suggested that nonspecific immobilization through one or more lysine residues close to or within the CDR2 region of the 11-1G10 VH domain was responsible for the reduced strength of the interaction with NC41 . These results emphasize the need to use site-specific immobilization strategies when accurate kinetic measurements are required .

Anal Biochem, 1997 Nov 1, 253(1), 37 - 45
Determination of the relative affinities of antibody fragments expressed in Escherichia coli by enzyme-linked immunosorbent assay; Watkins JD et al.; We have previously utilized an M13 phage expression system and codon-based mutagenesis to increase the avidity of the tumor-specific antibody, BR96, 65-fold (Yelton et al., 1995, J . Immunol . 155, 1994-2004) . Mutants with improved affinity were identified by screening phage-expressed antibodies on a carcinoma cell line . In this study we describe a more broadly applicable assay which permits rapid and quantitative comparison of affinities of related antibodies produced in an M13 phage expression system . BR96 variants displaying a range of affinities were expressed as soluble antibody fragments (Fabs) in the periplasmic space of bacteria and isolated from small-scale cultures grown in a 96-well format, yielding between 142 ng and 1.06 microg of Fab . Although the small-scale cultures expressed variable levels of Fab, the lower quantities were sufficient to saturate microtiter plates coated with a limiting amount of anti-human Fab antibody, resulting in the capture of uniform quantities of the Fab variants . The relative affinities of the variants were then compared by assessing binding to biotinylated antigen followed by detection with streptavidin-alkaline phosphatase conjugates . This approach permitted the direct comparison of the relative affinities of large numbers of antibody variants in a single step without multiple antibody dilutions . The assay is readily adaptable for screening phage-expressed antibody libraries against any biotinylated target and does not require purified antigen . For instance, the biotinylated BR96 antigen utilized in these studies was from a total cell extract prepared by labeling the surface of live tumor cells followed by detergent extraction . Thus, this approach may be applicable to the screening of antibody libraries against other cell surface antigens, such as transmembrane receptors, which are difficult to purify .

Biochem J, 1997 Oct 1, 327 ( Pt 1), 161 - 9
Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase; Chang AK et al.; Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine . The AHAS gene from Arabidopsis thaliana with part of the chloroplast transit sequence removed was cloned into the bacterial expression vector pT7-7 and expressed in the Escherichia coli strain BL21(DE3) . The expressed enzyme was purified by an extensive procedure involving (NH4)2SO4 fractionation followed by hydrophobic and anion-exchange chromatography . The purified enzyme appears as a single band on SDS/PAGE with a molecular mass of about 61 kDa . On gel filtration the enzyme is a dimer, migrating as a single peak with molecular masses of 109 and 113 kDa in the absence and presence of FAD respectively . Ion spray MS analysis yielded a mass of 63864 Da . The enzyme has optimum activity in the pH range 6.5-8.5 and exhibits absolute dependence on the three cofactors FAD, Mg2+ and thiamine diphosphate for activity . It displays negatively co-operative kinetics with respect to pyruvate concentration . A model was derived to explain the non-hyperbolic substrate-saturation curve, involving interaction between the active sites of the dimer . The Km for the first active site was found to be 8.01 +/- 0.66 mM; the Km for the second active site could not be accurately determined but was estimated to be approx . 100 mM . The enzyme is insensitive to valine, leucine and isoleucine but is strongly inhibited by the sulphonylurea herbicide, chlorsulphuron, and the imidazolinone herbicide, imazapyr . Inhibition by both herbicides exhibits slow-binding kinetics, whereas chlorsulphuron also shows tight-binding inhibition.

Biochem J, 1997 Oct 1, 327 ( Pt 1), 117 - 23
Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58; Chadwick MP et al.; The single-domain human trefoil proteins {pNR-2/pS2 and human intestinal trefoil factor (hITF)} have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain . The seventh does not form part of the trefoil domain and is located three residues from the C-terminus . The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE . pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not . Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds . To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2 . Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein . Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond . These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.

Vet Microbiol, 1997 Sep, 57(2-3), 119 - 33
Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle; Reddy JR et al.; The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli . The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells . The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA) and radio-immunoprecipitation (RIP) . The recombinant proteins isolated from bacteria showed antigenic properties when analyzed by ELISA and immunoblotting using BVDV antibodies . The recombinant proteins were then used in ELISA or IFA to detect BVDV infection by testing 54 independent bovine serum samples . The baculovirus-expressed BVDV protein was used as an ELISA and IFA antigen, and the bacteria-expressed proteins were used as ELISA antigens . BVDV-NADL-infected Madin-Darby bovine kidney (MDBK) cell monolayers served as a control antigen . Statistical analysis showed a high degree of correlation between the reactivity of recombinants and natural antigens in ELISA using bovine sera . The results of ELISA or IFA proved there is a high degree of correlation with the virus neutralization . In the comparative ELISA assays, the insect-cell-mediated expression revealed greater specificity and sensitivity than the bacterial expression or the natural BVDV antigens produced by cell cultures.

Nat Genet, 1997 Nov, 17(3), 298 - 304
Trinucleotide repeats affect DNA replication in vivo; Samadashwily GM et al.; (CGG)n.(CCG)n and (CTG)n.(CAG)n repeats of varying length were cloned into a bacterial plasmid, and the progression of the replication fork through these repeats was followed using electrophoretic analysis of replication intermediates . We observed stalling of the replication fork within repeated DNAs and found that this effect depends on repeat length, repeat orientation relative to the replication origin and the status of protein synthesis in a cell . Interruptions within repeated DNAs, similar to those observed in human genes, abolished the replication blockage . Our results suggest that the formation of unusual DNA structures by trinucleotide repeats in the lagging-strand template may account for the observed replication blockage and have relevance to repeat expansion in humans.

Blood, 1997 Nov 15, 90(10), 3874 - 83
Increased growth promoting but not mast cell degranulation potential of a covalent dimer of c-Kit ligand; Nocka KH et al.; The native form of soluble c-kit ligand (KL) is a noncovalent dimer . We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD . KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC . Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency . However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo . Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.

Biochemistry, 1997 Nov 4, 36(44), 13748 - 54
Inhibition of recombinant human mitochondrial and cytosolic aldehyde dehydrogenases by two candidates for the active metabolites of disulfiram; Lam JP et al.; We expressed recombinant human cytosolic (ALDH1, high Km) and mitochondrial aldehyde dehydrogenase (ALDH2, low Km) in Escherichia coli and purified the enzymes to homogeneity to examine the nature of inhibition of human ALDH by disulfiram, its confirmed metabolite S-methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide, and its proposed metabolite MeDTC sulfone . Disulfiram, MeDTC sulfoxide, and MeDTC sulfone, respectively, were potent inhibitors with IC50 values of 0.15 +/- 0.02 microM, 0.27 +/- 0.04 microM, and 0.12 +/- 0.02 microM for ALDH1, and 1.45 +/- 0.40 microM, 1.16 +/- 0.56, and 0.40 +/- 0.10 microM for ALDH2 . Extensive dialysis did not restore the activity of the inactivated enzyme, indicating irreversible inhibition . Both the esterase and dehydrogenase activities of ALDH2 were inhibited to the same extent by MeDTC sulfone and sulfoxide, suggesting that both catalytic sites are closely linked . The time course of inhibition of ALDH appeared to be first-order for both MeDTC sulfone and MeDTC sulfoxide . Kitz and Wilson plots of the half-life of inactivation versus 1/{inhibitor} indicated that the reactions between ALDH and inhibitors were bimolecular . The pseudobimolecular rate constants (k3/KI) for the ALDH-inhibitor reactions were 1 x 10(5), 1 x 10(4), 3 x 10(3), and 1 x 10(3) s-1 M-1 ALDH1-sulfone, ALDH1-sulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide, respectively . ALDH2 was not significantly protected from inactivation from either MeDTC sulfoxide or MeDTC sulfone by NAD alone, but high concentrations of NAD and acetaldehyde completely prevented inhibition . Since disulfiram is rapidly metabolized in vivo, it is believed that disulfiram is too short-lived to inhibit ALDH directly . The results of our study indicate that MeDTC sulfoxide and sulfone are potent inhibitors of human ALDH and are reasonable candidates for the proximal inhibitors of ALDH following disulfiram administration.

Biochemistry, 1997 Nov 4, 36(44), 13743 - 7
Expression and mutagenesis of mammalian cytosolic NADP+-specific isocitrate dehydrogenase; Jennings GT et al.; Rat liver cytosolic NADP+-specific isocitrate dehydrogenase (IDP2) was expressed in bacteria as a fusion protein with maltose binding protein (MBP) . High levels of expression were obtained . The fusion protein was purified from bacterial lysates by affinity chromatography with an amylose resin and found to be catalytically active . IDP2 was separated from MBP by cleavage with protease Xa and purified to homogeneity by FPLC anion-exchange chromatography . A specific activity of 56.3 units/mg and respective apparent Km values for dl-isocitrate and NADP+ of 9.7 +/- 2.9 microM and 11.5 +/- 0.2 microM were obtained for the purified enzyme . These values are similar to those previously reported for cytosolic isocitrate dehydrogenase isolated from a variety of tissues . Evolutionarily conserved arginine residues implicated in substrate binding were changed to glutamate residues using PCR based site-directed mutagenesis of the bacterial fusion plasmid . Mutant enzymes containing residue changes of R100E, R109E, R119E, or R132E were expressed, purified, and characterized by initial rate kinetic analyses . The R119E and R109E mutant enzymes exhibited respective 15- and 31-fold increases in Km values for dl-isocitrate relative to the wild-type enzyme . In contrast, Km values for NADP+ were, respectively, unchanged and increased 9-fold . The most significant reductions in kcat/Km values were obtained for the R100E, R109E, and R132E enzymes . These results suggest that substrate binding residues are highly conserved between bacterial and mammalian enzymes despite low overall homology.

Biochemistry, 1997 Nov 4, 36(44), 13736 - 42
A conserved glutamic acid in helix VI of cytochrome bo3 influences a key step in oxygen reduction; Watmough NJ et al.; We have compared the reactions with dioxygen of wild-type cytochrome bo3 and a mutant in which a conserved glutamic acid at position-286 of subunit I has been changed to an alanine . Flow-flash experiments reveal that oxygen binding and the rate of heme-heme electron transfer are unaffected by the mutation . Reaction of the fully (3-electron) reduced mutant cytochrome bo3 with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen {Puustinen, A., et al . (1996) Proc . Natl . Acad . Sci . U.S.A . 93, 1545-1548} . These results confirm that proton uptake is important in controlling the later stages of dioxygen reduction in heme-copper oxidases and show that E286 is an important component of the channel that delivers these protons to the active site.

Biochemistry, 1997 Nov 4, 36(44), 13700 - 9
Conformational analysis of Escherichia coli 30S ribosomes containing the single-base mutations G530U, U1498G, G1401C, and C1501G and the double-base mutation G1401C/C1501G; Moine H et al.; Biochemical and genetic studies have pointed out the importance of several sites in 16S ribosomal RNA of Escherichia coli in the decoding process . These sites consist of the core of the decoding center (1400/1500 region) and two other segments (530 and 1050/1200 regions) . To detect a possible structural link between these functionally related regions, we analyzed their sensitivity to conformational changes induced by mutations which are located in each of these regions and are known to affect the decoding process . The conformations of five segments of 16S rRNA (1-106, 406-569, 780-978, 997-1247, and 1334-1519) were analyzed by chemical probing of 30S ribosomes containing the following mutations: G530U, U1498G, G1401C, C1501G, and G1401C/C1501G . Ribosomes reconstituted with natural wild-type 16S RNA showed only minor conformational differences with respect to ribosomes isolated from cells . When 16S RNA made in vitro replaced natural 16S RNA, a slightly looser conformation of the central core region was found . Mutant ribosomes made by reconstitution with mutant 16S RNA made in vitro showed conformational effects which were in all cases localized to the region of secondary structure surrounding the site of mutation . Although the core of the decoding center (1400/1500 region) and the two other sites (530 and 1050/1200 regions) participating in the decoding function have been functionally linked, our data indicate that they are structurally independent . They also provide evidence for an unusual structure of the 1400/1500 decoding center, possibly involving noncanonical interactions . Furthermore, the absence of any conformational effect induced by the G530U mutation except at the site of mutation itself points to its direct, as opposed to indirect, involvement in the decoding function of the ribosome.

Biochemistry, 1997 Nov 4, 36(44), 13693 - 9
A single amino acid substitution in ribonucleolytic toxin restrictocin abolishes its specific substrate recognition activity; Nayak SK et al.; Restrictocin is a small basic protein produced by the fungus Aspergillus restrictus . It potently inhibits protein synthesis in eukaryotic cells by specifically cleaving a single phosphodiester bond in 28S rRNA . A histidine residue at position 49 in restrictocin has been implicated in its active site . A mutant of restrictocin in which the histidine at position 49 was changed to an alanine was constructed by site-directed mutagenesis, and the protein was expressed in Escherichia coli . The mutant and the wild type proteins were found to be structurally identical . Unlike restrictocin, the restrictocin H49A mutant did not cleave the ribosomal RNA specifically at the target phosphodiester bond; instead, it extensively degraded the RNA substrate with altered specificity . The mutant exhibited a high ribonuclease activity compared to restrictocin on yeast tRNA, and poly(U) and poly(C) . The mutant also poorly inhibited protein synthesis in eukaryotic cells as well as in a cell free system . We therefore propose that histidine 49 of restrictocin is not involved per se in the enzymatic activity; however, it does play a crucial role in the specific recognition of the target sequence by restrictocin.

Biochemistry, 1997 Nov 4, 36(44), 13688 - 92
Interaction between residues Glu269 (helix VIII) and His322 (helix X) of the lactose permease of Escherichia coli is essential for substrate binding; He MM et al.; Site-directed and Cys-scanning mutagenesis of the lactose permease of Escherichia coli reveals that as few as four residues--Glu269 (helix VIII), Arg302 (helix IV), His322 (helix X), and Glu325 (helix X)--are irreplaceable for coupling substrate and H+ translocation . Interestingly, the four residues are in close physical proximity, Glu269 interacting with His322 and Arg302 with Glu325 . In addition, the substrate translocation pathway is located close to the four residues at the interface between helices V and VIII . To investigate the importance of the four residues and their interactions for substrate binding, mutation Glu269-->Asp, Glu269-->Gln, Arg302-->Ala, Arg302-->Lys, His322-->Ala, His322-->Phe, Glu325-->Asp, or Glu325-->Gln was introduced into single-Cys148 permease, where the reactivity of Cys with 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) is blocked by binding of substrate . The double mutants were purified, and the rates of MIANS labeling were measured in the absence or presence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG), lactose, or galactose at various concentrations . Remarkably, substrate binding by the Glu269 or His322 mutants is abolished or decreased dramatically, while binding by the Arg302 or Glu325 mutants is not altered . The observations are consistent with the notion that the interaction between Glu269 and His322 stabilizes the interface between helices V and VIII and thereby leads to binding of substrate.

Biochemistry, 1997 Nov 4, 36(44), 13682 - 7
Arginine 302 (helix IX) in the lactose permease of Escherichia coli is in close proximity to glutamate 269 (helix VIII) as well as glutamate 325; He MM et al.; By using a variety of biochemical and biophysical approaches, a helix packing model for the lactose permease of Escherichia coli has been proposed in which the four residues that are irreplaceable with respect to coupling are paired--Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix XI) with Glu325 (helix X) . In addition, the substrate translocation pathway is located at the interface between helices V and VIII, which is in close vicinity to the four essential residues . Based on this structural information and functional studies of mutants in the four irreplaceable residues, a molecular mechanism for energy coupling in the permease has been proposed {Kaback, H . R . (1997) Pro.c Natl . Acad . Sci . U.S.A . 94, 5539} . The principle idea of this model is that Arg302 interacts with either Glu325 or Glu269 during turnover . Evidence that Arg302 is in close proximity with Glu325 has been presented {Jung, K., Jung, H., Wu, J., Prive, G . G., & Kaback, H . R . (1993) Biochemistry 32, 12273; He, M . M., Voss, J., Hubbell, W . L., & Kaback, H . R . (1995) Biochemistry 34, 15667}; however, the proximity of Arg302 to Glu269 has not been examined . In this report, it is shown by two methods that Arg302 is also close to Glu269: (i) permease with Glu269-->His, Arg302-->His, and His322-->Phe binds Mn2+ with high affinity at pH 7.5, but not at pH 5.5; and (ii) site-directed spin-labeling of the double Cys mutant Glu269-->Cys/Arg302-->Cys exhibits spin-spin interaction with an interspin distance of about 14-16 A . In addition, the spin-spin interaction is stronger and interspin distance shorter after the permease is reconstituted into proteoliposomes . Taken as a whole, the data are consistent with the idea that Arg302 may interact with either Glu325 or Glu269 during turnover.

J Biochem (Tokyo), 1997 Jun, 121(6), 1102 - 6
Purification and characteristics of recombinant mouse metallothionein-I from Escherichia coli; Xiong Y et al.; The mouse metallothionein-I (mMT-I) cDNA was amplified by polymerase chain reaction (PCR), inserted into vector pGEX-4T-1, and expressed in Escherichia coli as a carboxyl terminal extension of the 26-kDa glutathione-S-transferase (GST) . Analyzed by SDS-PAGE, the amount of the expressed fusion protein GST-MT was over 50% of total cellular proteins . After the fusion protein had been digested with thrombin on a Glutathione-Sepharose 4B affinity chromatography column, recombinant mMT-I was purified by gel filtration on Sephadex G50 . The results of molecular mass, amino acid composition and sequence of 10 amino acids at the N-terminus of the recombinant mMT-I demonstrate that the purified protein is the one we desired . The ratios of metal:protein and thiol:protein are the same as those of wild-type MT . The half-dissociation pHs of Cd, Cu, and Zn from recombinant mMT-I were 3.57, 1.40, and 5.20, respectively, which are in agreement with those from native rabbit MT-I . The ultraviolet absorbance and circular dichroism (CD) spectra at pH 8.0 and pH 2.0 were all similar to those of native MT, indicating that they have the same metal-thiolate structure even though six amino acid residues have been added at the N-terminus of the recombinant protein.

J Biochem (Tokyo), 1997 Jun, 121(6), 1002 - 9
The two lectin domains of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans have different binding properties . Studies with recombinant protein; Arata Y et al.; Some properties of recombinant proteins derived from the 32-kDa galectin isolated from the nematode Caenorhabditis elegans, which lectin is composed of two tandemly repeated homologous domains {Hirabayashi et al . (1992) J . Biol . Chem . 267, 15485}, were studied in order to elucidate the function of this unique polypeptide architecture . We expressed the whole molecule (N32), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) in Escherichia coli using the expression vector pET21a . All of the recombinant proteins were bound by asialofetuin-Sepharose . CD spectra of the recombinant proteins indicated all of them to be rich in beta-structure and properly refolded . Gel filtration on an HPLC column suggested that all of them existed as monomers . Neither Nh nor Ch seemed to form dimers, in contrast to vertebrate proto-type galectins . Only N32 showed hemagglutination activity towards trypsinized rabbit erythrocytes . Comparison of the affinity of N32, Nh, and Ch for asialofetuin-Sepharose by frontal affinity chromatography {Kasai et al . (1986) J . Chromatogr . 376, 33} showed that Ch has 7-fold weaker affinity than N32, and Nh proved to have still weaker affinity . Since the Asn residue in the CRD (carbohydrate recognition domain), which is conserved in all other galectins, is substituted by Ser in the case of Nh, these data suggest that the two CRDs in this tandem-repeat galectin have different sugar binding properties and that the 32-kDa galectin may serve as a heterobifunctional crosslinker.

Aliment Pharmacol Ther, 1997 Oct, 11(5), 853 - 8
Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis; Kruis W et al.; BACKGROUND: Aminosalicylates are used as standard treatment for maintaining remission in ulcerative colitis . As yet, there is no other existing alternative with proven efficacy . In light of the hypothesis that the intestinal environment may contribute to the pathophysiology of ulcerative colitis, a trial was conducted to test the effects of probiotic treatment with an oral preparation of non-pathogenic E . coli . METHODS: A total of 120 patients with inactive ulcerative colitis were included in a double-blind, double-dummy study comparing mesalazine 500 mg t.d.s . to an oral preparation of viable E . coli strain Nissle (Serotype 06: K5: H1) for 12 weeks with regard to their efficacy in preventing a relapse of the disease . Study objectives were to assess the equivalence of the clinical activity index (CAI) under the two treatment modalities and to compare relapse rates, relapse-free times and global assessment . RESULTS: The start and end scores of the CAI demonstrated no significant difference (P = 0.12) between the two treatment groups . Relapse rates were 11.3% under mesalazine and 16.0% under E . coli Nissle 1917 (N.S.) . Life table analysis showed a relapse-free time of 103 +/- 4 days for mesalazine and 106 +/- 5 days for E . coli Nissle 1917 (N.S.) . Global assessment was similar for both groups . Tolerability to the treatment was excellent and did not differ . No serious adverse events were reported . CONCLUSIONS: From the results of this preliminary study, probiotic treatment appears to offer another option for maintenance therapy of ulcerative colitis . Additional support is provided for the hypothesis of a pathophysiological role for the intestinal environment in ulcerative colitis.

Microbiology, 1997 Oct, 143 ( Pt 10), 3101 - 10
Transcriptional control of several aerobically induced cytochrome structural genes in Rhodobacter sphaeroides; Flory JE et al.; To decipher how the synthesis of energy-transducing enzymes responds to environmental cues, the response of three Rhodobacter sphaeroides aerobic cytochrome gene promoters was analysed under different conditions . Two of these promoters are upstream of structural genes (ctaD and coxII) for individual subunits of the cytochrome aa3 respiratory complex . The third promoter is that for the cycFG operon, which encodes two c-type cytochromes of unknown function, cytochrome c554 and CycG . Primer extension analysis identified a single oxygen-responsive transcription start site for each gene . Utilizing operon fusions to Escherichia coli lacZ as a measure of promoter activity, transcription from the ctaD, coxII and cycFG promoters was approximately twofold higher when cells were grown at high (30%) oxygen tensions than under low (2%) oxygen or anaerobic (photosynthetic) conditions . Analysis of promoter function using specific host mutations indicated that loss of the R . sphaeroides FNR homologue, FnrL, causes a small, but reproducible, increase in cycFG and coxII transcription when cells are grown at 2% oxygen . However, neither the delta FnrL mutation nor alterations in sequences related to a consensus target site for the E . coli FNR protein increased function of any of these three promoters to that seen under aerobic conditions in wild-type cells . From this we conclude that FnrL is not solely responsible for reduced transcription of these three aerobic cytochrome genes under low oxygen or anaerobic conditions . When activity of these three promoters was monitored after cells were shifted from anaerobic (photosynthetic) conditions to a 30% oxygen atmosphere, it took several cell doublings for LacZ levels to increase to those found in steady-state 30% oxygen cultures . From these results, it appears that activity of these promoters is also regulated by a stable molecule whose synthesis or function responds slowly to the presence of high oxygen tensions.

J Pharmacol Exp Ther, 1997 Nov, 283(2), 918 - 24
Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production by dexanabinol (HU-211), a nonpsychotropic cannabinoid; Gallily R et al.; Dexanabinol, HU-211, a synthetic cannabinoid devoid of psychotropic effects, improves neurological outcome in models of brain trauma, ischemia and meningitis . Recently, HU-211 was found to inhibit brain tumor necrosis factor (TNFalpha) production after head injury . In the present study, we demonstrate the ability of HU-211 to suppress TNFalpha production and to rescue mice and rats from endotoxic shock after LPS (Escherichia coli 055:B5) inoculation . In BALB/c mice, a dose of 10 mg/kg LPS, injected i.p., caused 57% and 100% mortality, at 24 and 48 hr, respectively . HU-211, administered i.p . 30 min before lipopolysaccharide (LPS), reduced lethality to 9 and 67% at these time points (P < .05) . When coinjected with D-galactoseamine (i.p.), LPS was 100% lethal within 24 hr, whereas eight hourly injections of HU-211 caused mortality of C57BL/6 mice to drop to 10% (P < .001) . Administration of LPS to Sprague-Dawley rats resulted in a 30% reduction in the mean arterial blood pressure within 30 min, which persisted for 3 hr . HU-211, given 2 to 3 min before LPS, completely abolished the typical hypotensive response . Furthermore, the drug also markedly suppressed in vitro TNFalpha production and nitric oxide generation (by >90%) by both murine peritoneal macrophages and rat alveolar macrophage cell line exposed to LPS . HU-211 may, therefore, have therapeutic implications in the treatment of TNFalpha-mediated pathologies.

J Biol Chem, 1997 Nov 7, 272(45), 28652 - 9
Cloning, expression, and characterization of two manganese superoxide dismutases from Caenorhabditis elegans; Hunter T et al.; Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans . Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same . The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous . Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides . Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts are trans-spliced to the SL-1 leader sequence . The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts . The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I . Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels in Escherichia coli cells deficient in cytosolic superoxide dismutases . Both proteins were shown to be active in E . coli, providing similar protection against methyl viologen-induced oxidative stress . The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.

J Biol Chem, 1997 Nov 7, 272(45), 28638 - 45
UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine-containing peptides and eukaryotic cysteine proteinases; Mehta DP et al.; Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins . Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae . GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs . We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues . Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them . Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency . Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase . CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM . SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all . Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM . Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs . This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.

J Biol Chem, 1997 Nov 7, 272(45), 28518 - 22
Echovirus 1 interaction with the human very late antigen-2 (integrin alpha2beta1) I domain . Identification of two independent virus contact sites distinct from the metal ion-dependent adhesion site; King SL et al.; The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1 . Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the alpha2 subunit of the VLA-2 alpha2beta1 heterodimer . Although murine VLA-2 interacts with collagen, it does not bind virus . We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199-201 and 212-216, independently involved in virus attachment . These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen . Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding . These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.

J Biol Chem, 1997 Nov 7, 272(45), 28431 - 7
A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif; Leon DA et al.; The RIalpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus . Based on resistance to proteolysis and expression as a discrete domain in Escherichia coli, this motif is defined as residues 12-61 . This motif is chemically, kinetically, and thermally stable . The two endogenous interchain disulfide bonds between Cys16 and Cys37 in RIalpha are extremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell . The disulfide bonds were present in recombinant RIalpha as well as when the dimerization domain alone was expressed in E . coli, emphasizing the unusual stability of this motif and the disulfide bonds . Although 100 mM dithiothreitol was sufficient to reduce the disulfide bonds, it did not abolish dimerization . In addition, a stable dimer also still formed when Cys37 was replaced with His, confirming unambiguously the original antiparallel alignment of the disulfide bonds . Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization . Circular dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix . Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed . A new alignment of the dimerization domains of RI, RII, and cGMP-dependent protein kinase elucidates fundamental similarities as well as significant differences among these three domains.

J Biol Chem, 1997 Nov 7, 272(45), 28391 - 7
Cell envelope signaling in Escherichia coli . Ligand binding to the ferrichrome-iron receptor fhua promotes interaction with the energy-transducing protein TonB; Moeck GS et al.; The ferrichrome-iron receptor of Escherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25 . In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB . In an E . coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker . The complex included both proteins; it was absent from strains of E . coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies . These results indicate that, in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB . An in vitro system was established with which to examine the FhuA-TonB interaction . Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA . Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB . Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA . These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane.

J Biol Chem, 1997 Nov 7, 272(45), 28381 - 90
The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis; Unkles SE et al.; The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway . The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC . Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type . The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E . coli moaC product, with both E . coli products encoded by different cistrons . In the wild type, precursor Z levels are 3-4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway . Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein . In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase . More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor . This supports the proposition that these residues play an essential catalytic role . An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only . A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function.

J Biol Chem, 1997 Nov 7, 272(45), 28368 - 72
A protein phosphatase-1-binding motif identified by the panning of a random peptide display library; Zhao S et al.; An unusually large number of regulatory or targeting proteins that bind to the catalytic subunit of protein phosphatase-1 have been recently reported . This can be explained by their possession of a common protein motif that interacts with a binding site on protein phosphatase-1 . The existence of such a motif was established by the panning of a random peptide library in which peptide sequences are displayed on the Escherichia coli bacterial flagellin protein for bacteria that bound to protein phosphatase-1 . There were 79 isolates containing 46 unique sequences with the conserved motif VXF or VXW, where X was most frequently His or Arg . In addition, this sequence was commonly preceded by 2-5 basic residues and followed by 1 acidic residue . This study demonstrates that binding to protein phosphatase-1 can be conferred to a protein by the presentation of a peptide motif on a surface loop . This binding motif is found in a number of protein phosphatase-1-binding proteins.

J Biol Chem, 1997 Nov 7, 272(45), 28267 - 73
Molecular heterogeneity of phospholipase D (PLD) . Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by polyphosphoinositides and calcium; Qin W et al.; Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, vesicle trafficking, and membrane metabolism . This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLDgamma, and the regulation of PLDgamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ . The PLDgamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pI of 6.9 . PLDgamma shares a 66% amino acid sequence identity with PLDbeta, but only a 41% identity with PLDalpha . A potential N-terminal myristoylation site is found in PLDgamma, but not in PLDalpha and -beta . Catalytically active PLDgamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides . Both PLDgamma and -beta are most active at microM Ca2+ concentrations, whereas the optimal PLDalpha activity requires mM Ca2+ concentrations . Binding studies showed that the PLDs bound PIP2 in the order of PLDbeta > PLDgamma > PLDalpha . This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site . The binding of {3H}PIP2 was saturable and could be competitively decreased by addition of unlabeled PIP2 . Neomycin inhibited the activities of PLDgamma and -beta, but not PLDalpha . These results demonstrate that PLD is encoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLDgamma and -beta, but not PLDalpha . The different structural and biochemical properties suggest that PLDalpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.

J Biol Chem, 1997 Nov 7, 272(45), 28175 - 8
Promoter escape by RNA polymerase II . Formation of an escape-competent transcriptional intermediate is a prerequisite for exit of polymerase from the promoter; Dvir A et al.; Shortly after initiating promoter-specific transcription in vitro, mammalian RNA polymerase II becomes highly susceptible to arrest in a promoter-proximal region 9-13 base pairs downstream of the transcriptional start site (Dvir, A., Conaway, R . C., and Conaway, J . W . (1996) J . Biol . Chem . 271, 23352-23356) . Arrest by polymerase in this region is suppressed by TFIIH in an ATP-dependent reaction (Dvir, A., Conaway, R . C., and Conaway, J . W . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 9006-9010) . In this report, we present evidence that, in addition to TFIIH and an ATP cofactor, efficient transcription by RNA polymerase II through this promoter-proximal region requires formation of an "escape-competent" transcriptional intermediate . Formation of this intermediate requires template DNA 40-50 base pairs downstream of the transcriptional start site . This requirement for downstream DNA is transient, since template DNA downstream of +40 is dispensable for assembly of the preinitiation complex, for initiation and synthesis of the first 10-12 phosphodiester bonds of nascent transcripts and for further extension of transcripts longer than approximately 14 nucleotides . Thus, promoter escape requires that the RNA polymerase II transcription complex undergoes a critical structural transition, likely driven by interaction of one or more components of the transcriptional machinery with template DNA 40-50 base pairs downstream of the transcriptional start site.

Genes Dev, 1997 Nov 1, 11(21), 2780 - 9
Casein kinase II regulation of yeast TFIIIB is mediated by the TATA-binding protein; Ghavidel A et al.; The highly conserved protein kinase casein kinase II (CKII) is required for efficient Pol III transcription of the tRNA and 5S rRNA genes in Saccharomyces cerevisiae . Using purified factors from wild-type cells to complement transcription extracts from a conditional lethal mutant of CKII we show that TFIIIB is the CKII-responsive component of the Pol III transcription machinery . Dephosphorylation of TFIIIB eliminated its ability to complement CKII-depleted extract, and a single TFIIIB subunit, the TATA-binding protein (TBP), is a preferred substrate of CKII in vitro . Recombinant TBP purified from Escherichia coli is phosphorylated efficiently by CKII and, in the presence of a limiting amount of CKII, is able to substantially rescue transcription in CKII-deficient extract . Our results establish that TBP is a key component of the pathway linking CKII activity and Pol III transcription and suggest that TBP is the target of a CKII-mediated regulatory mechanism that can modulate Pol III transcription.

Infect Immun, 1997 Nov, 65(11), 4494 - 501
Binding of diarrheagenic Escherichia coli to 32- to 33-kilodalton human intestinal brush border proteins; Manjarrez-Hernandez A et al.; We have detected human intestinal brush border proteins to which Escherichia coli strains adhere by means of a blotting-nitrocellulose method in which the binding of radiolabeled bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated intestinal cell membranes was evaluated . The brush border fraction contained several polypeptides that bound only adherent E . coli strains . The most prominent and consistent of these proteins had apparent molecular masses of 32 to 33 kDa . Additional polypeptides ranging from 50 to 70, from 105 to 130, and from 180 to 200 kDa were also recognized by adherent E . coli strains, although with less intensity (in accordance with the number of bound bacteria to these polypeptides) . Independently of the pattern of adherence (localized {LA}, diffuse {DA}, or aggregative {AggA}) all HEp-2-adhering strains recognized, with different intensities, the 32- to 33-kDa brush border proteins, whereas nonadhesive strains did not . The relative avidity of an LA strain to bind to the 32- to 33-kDa proteins was approximately seven- and sixfold higher than the binding of strains with aggregative and diffuse adherence, respectively . Thus, it is reasonable to think that LA, DA, and AggA strains have a common adhesin that mediates binding to the 32- to 33-kDa bands . Inhibition experiments using HEp-2 cells demonstrated that isolated 32- to 33-kDa proteins or specific antiserum blocked preferentially bacterial adherence of the LA pattern . Delipidization and protein digestion of the human brush borders confirmed that E . coli bound to structures of a proteinaceous nature . Deglycosylation studies and sodium meta-periodate oxidation of the intestinal cell membranes decreased bacterial binding activity significantly, indicating that E . coli bound to carbohydrate moieties in the glycoproteins . These results suggest that binding of E . coli strains, mainly of the LA phenotype, to the 32- to 33-kDa proteins could play a role in colonization through adherence to the intestinal mucosa.

Infect Immun, 1997 Nov, 65(11), 4384 - 8
Borrelia burgdorferi induces chemokines in human monocytes; Sprenger H et al.; Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response . To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis . Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus . The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis . The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h . Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP-1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS . Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils . Thus, B . burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease.

J Bacteriol, 1997 Nov, 179(21), 6862 - 4
SigmaE is an essential sigma factor in Escherichia coli; De Las Penas A et al.; SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli . SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C . We present evidence that sigmaE is an essential sigma factor at all temperatures . Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation.

J Bacteriol, 1997 Nov, 179(21), 6858 - 61
Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli; Ebel W et al.; Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon+ cells, increased 38-fold in lon+ cells carrying a Tn10::delta kan insertion mapping to 24 min on the E . coli chromosome . Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::delta kan insertion . Sequencing of both sides of the Tn10::delta kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs) . A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.

J Bacteriol, 1997 Nov, 179(21), 6831 - 6
Catalase-peroxidase of Caulobacter crescentus: function and role in stationary-phase survival; Steinman HM et al.; Caulobacter crescentus is an obligate aerobe which is exposed to high concentrations of photosynthetic oxygen and low levels of nutrients in its aquatic environment . Physiological studies of oxidative and starvation stresses in C . crescentus were undertaken through a study of lacZ fusion and null mutant strains constructed from the cloned 5' end of katG, encoding a catalase-peroxidase . The katG gene was shown to be solely responsible for catalase and peroxidase activity in C . crescentus . Like the katG of Escherichia coli, C . crescentus katG is induced by hydrogen peroxide and is important in sustaining the exponential growth rate . However, dramatic differences are seen in growth stage induction . E . coli KatE catalase and KatG catalase-peroxidase activities are induced 15- to 20-fold during exponential growth and then approximately halved in the stationary phase . In contrast, C . crescentus KatG activity is constant throughout exponential growth and is induced 50-fold in the stationary phase . Moreover, the survival of a C . crescentus katG null mutant is reduced by more than 3 orders of magnitude after 24 h in stationary phase and more than 6 orders of magnitude after 48 h, a phenotype not seen for E . coli katE and katG null mutants . These results indicate a major role for C . crescentus catalase-peroxidase in stationary-phase survival and raise questions about whether the peroxidatic activity as well as the protective catalatic activity of the dual-function enzyme is important in the response to starvation stress.

J Bacteriol, 1997 Nov, 179(21), 6807 - 15
The Helicobacter pylori genome is modified at CATG by the product of hpyIM; Xu Q et al.; To understand mechanisms of DNA methylation in Helicobacter pylori, a human pathogen associated with peptic ulcer disease and gastric adenocarcinoma, we cloned a putative DNA methyltransferase gene, hpyIM . This gene contains a 990-bp open reading frame encoding a 329-amino-acid protein, M.HpyI . Sequence analysis revealed that M.HpyI was closely related to CATG-recognizing adenine DNA methyltransferases, including M.NlaIII in N . lactamica . hpyIM was present in all H . pylori strains tested . DNA from wild-type H . pylori strains was resistant to digestion by SphI and NlaIII, which recognize DNA at sites containing CATG, whereas their isogenic hpyIM mutants were susceptible, indicating lack of modification . Overexpression of hpyIM in Escherichia coli rendered DNA from these cells resistant to NlaIII digestion, confirming the role of hpyIM in modifying CATG sites . We conclude that hpyIM encodes a DNA methyltransferase, M.HpyI, that is well conserved among diverse H . pylori strains and that modifies H . pylori genomes at CATG sites.

J Bacteriol, 1997 Nov, 179(21), 6721 - 8
Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme; Kim DR et al.; Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain . Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site . Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold . The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time . With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E . coli sequence by inspection of conserved acidic amino acids . The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria . The E . coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.

J Bacteriol, 1997 Nov, 179(21), 6714 - 20
High- and low-abundance chemoreceptors in Escherichia coli: differential activities associated with closely related cytoplasmic domains; Feng X et al.; In Escherichia coli, two high-abundance chemoreceptors are present in cellular dosages approximately ten-fold greater than two low-abundance receptors . In the absence of high-abundance receptors, cells exhibit an abnormally low tumble frequency and the ability of the remaining receptors to mediate directed migration in spatial gradients is substantially compromised . We found that increasing the cellular amount of the low-abundance receptor Trg over a range of dosages did not alleviate these defects and thus concluded that high- and low-abundance receptors are distinguished not simply by their different dosages in a wild-type cell but also by an inherent difference in activity . By creating hybrids of the low-abundance receptor Trg and the high-abundance receptor Tsr, we investigated the possibility that this inherent difference could be localized to a specific receptor domain and found that the cytoplasmic domain of the high-abundance receptor Tsr conferred the essential features of that receptor class on the low-abundance receptor Trg, even though it is in this domain that residue identity between the two receptors is substantially conserved.

J Bacteriol, 1997 Nov, 179(21), 6705 - 13
IF3-mediated suppression of a GUA initiation codon mutation in the recJ gene of Escherichia coli; Haggerty TJ et al.; A mutational change of the initiation codon to GUA was found to reduce, but not abolish, expression of the recJ gene of Escherichia coli . Specific mutations in translational initiation factor IF3 have been isolated as second-site suppressors of this GUA initiation codon mutation . One of these, infC135, with an arginine-to-proline change at amino acid 131, completely restores a wild-type phenotype to recJ GUA initiation codon mutants and acts in a semidominant fashion . The infC135 mutation increased expression of RecJ from the GUA mutant but had no effect on the normal GUG start . The infC135 mutation also abolished autoregulation of IF3 in cis and in trans . The behavior of this IF3 mutant suggests that it has specifically lost its ability to abort initiation from poor initiation codons such as GUA of recJ and the AUU of infC . Because of the impact of IF3 on recJ, a recombination and repair gene, this role of IF3 must be general and not restricted to translation genes . The dominance of infC135 suggests that the other functions of IF3, for instance its ability to bind to 30S ribosomes, must remain intact . Although the ability to discriminate among initiation codons has been lost in the infC135 mutant, translational initiation was still restricted to the normal initiation site in recJ, even in the presence of a closely juxtaposed alternative initiation codon . Because the recJ gene lacks a canonical Shine-Dalgarno sequence, other unknown features of the mRNA must serve to specify the initiation site.

J Bacteriol, 1997 Nov, 179(21), 6665 - 73
Regulation of upp expression in Escherichia coli by UTP-sensitive selection of transcriptional start sites coupled with UTP-dependent reiterative transcription; Tu AH et al.; Expression of the upp gene of Escherichia coli, which encodes the pyrimidine salvage enzyme uracil phosphoribosyltransferase, is negatively regulated by pyrimidine availability . In this study, we demonstrate that this regulation occurs mainly by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be productively elongated . The upp initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand) . Transcription is initiated primarily at the first two bases in this sequence, designated G6 and A7 (counting from the promoter -10 region) . High intracellular levels of UTP favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive nucleotide addition) within the run of T residues in the initially transcribed region . The resulting AUUUUn (where n = 1 to >50) transcripts are not extended to include downstream upp sequences . In contrast, low intracellular levels of UTP strongly favor initiation at position G6, which results in transcripts that generally do not engage in reiterative transcription and thus can be normally elongated . This mechanism ensures that high levels of uracil phosphoribosyltransferase are produced only under conditions of pyrimidine limitation . The mechanisms that account for UTP-sensitive start site selection and different fates of upp transcripts, as well as the general use of UTP-dependent reiterative transcription in gene regulation, are discussed in detail.

J Bacteriol, 1997 Nov, 179(21), 6649 - 56
Deletion of the N-terminal region of the AREA protein is correlated with a derepressed phenotype with respect to nitrogen metabolite repression; Lamb HK et al.; The entire areA gene and a truncated version lacking the sequence encoding the N-terminal 389 amino acids were expressed from the qutE promoter and terminator in an Aspergillus nidulans strain with the endogenous areA gene deleted . This expression system was used to decouple the effects of transcription regulation and mRNA stability mediated by the native promoter and terminator from any posttranslational modulation of AREA activity . Both the full-length AREA protein and the truncated form were able to function in the deletion strain, conferring the ability to use alternate nitrogen sources . Transformants containing the entire areA gene had a repressible phenotype with respect to nitrogen metabolite repression, whereas those containing the truncated form of the areA gene had a derepressed phenotype . The truncated areA gene was expressed in an A . nidulans strain containing a normally regulated wild-type areA gene, and transformants displayed a quinate-inducible nitrogen metabolite derepressed phenotype . Northern blot analysis of transformed strains showed that areA-specific mRNAs of the expected sizes were being produced . The truncated AREA protein was overproduced in Escherichia coli as a fusion protein and purified to homogeneity by a single-step immobilized metal affinity chromatography, and the purified protein was shown to bind specifically to the niaD promoter . Revised sequences of the 5' region of the areA gene and the entire meaB gene are reported.

J Bacteriol, 1997 Nov, 179(21), 6618 - 25
Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS; Donato GM et al.; The H-NS protein is a major component of the Escherichia coli nucleoid . Mutations in hns, the gene encoding H-NS, have pleiotropic effects on the cell altering both the expression of a variety of unlinked genes and the inversion rate of the DNA element containing the fimA promoter . We investigated the interaction between H-NS and fimB, the gene encoding the bidirectional recombinase that catalyzes fimA promoter flipping . In beta-galactosidase assays, we found that fimB expression increased approximately fivefold in an hns2-tetR insertion mutant . In gel mobility shift assays with purified H-NS, we have also shown that H-NS bound directly and cooperatively to the fimB promoter region with greater affinity than for any other known H-NS-regulated gene . Furthermore, this high-affinity interaction resulted in a promoter-specific inhibition of fimB transcription . The addition of purified H-NS to an in vitro transcription system yielded a fivefold or greater reduction in fimB-specific mRNA production . However, the marked increase in cellular FimB levels in the absence of H-NS was not the primary cause of the mutant rapid inversion phenotype . These results are discussed in regard to both H-NS as a transcriptional repressor of fimB expression and its role in regulating type 1 pilus promoter inversion.

J Bacteriol, 1997 Nov, 179(21), 6602 - 8
Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin; Rietsch A et al.; The Escherichia coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase . For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form . Here we present evidence that, in wild-type cells, these two cysteines are reduced . Further, we show that a pathway involving the cytoplasmic proteins thioredoxin reductase and thioredoxin and the cytoplasmic membrane protein DsbD is responsible for the reduction of these cysteines . Thus, reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC . This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway . The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds . Analysis of the activity of mutant forms of DsbC in which either or both of these cysteines have been altered further supports the role of DsbC as a disulfide bond isomerase.

J Bacteriol, 1997 Nov, 179(21), 6573 - 80
Thermosensing properties of mutant aspartate chemoreceptors with methyl-accepting sites replaced singly or multiply by alanine; Nishiyama S et al.; The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modification of its four methylation sites (Gln295, Glu302, Gln309, and Glu491) . Without posttranslational deamidation, Tar has no thermosensing ability . When Gln295 and Gln309 are deamidated to Glu, the unmethylated and heavily methylated forms function as warm and cold sensors, respectively . In this study, we carried out alanine-scanning mutagenesis of the methylation sites . Although alanine substitutions influenced the signaling bias and the methylation level, all of the mutants retained aspartate-sensing function . Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function . In the posttranslational modification-defective background, some of the alanine substitutions restored thermosensing ability . Warm sensors were found among mutants retaining two glutamate residues, and cold sensors were found among those with one or no glutamate residue . This result suggests that the negative charge at the methylation sites is one factor that determines thermosensor phenotypes, although the size and shape of the side chain may also be important . The warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes were found . Thus, the different thermosensing phenotypes that result from covalent modification of the methylation sites may reflect distinct structural states . Broader implications for the thermosensing mechanism are also discussed.

Bioseparation, 1996, 6(6), 361 - 72
Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli; Wong HH et al.; The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photoSedimentation (CDS) . The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated . Increasing the normalised centrifuge feedrate (Q/sigma) from 1.32 x 10(-9) m s-1 to 3.97 x 10(-9) m s-1 leads to a 36% increase in inclusion body paste purity . Purity may also be improved by repeated homogenisation . Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered . At a normalised centrifuge feedrate of 2.65 x 10(-9) m s-1, increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58% . Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study . The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.16 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris . This is the first accurate experimentally-determined grade efficiency curve for cell debris . Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g . spherical PVA particles) . The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

Microbiol Res, 1997 Sep, 152(3), 251 - 5
Differential amplification efficiency of pMB1 and p15A (ColE1-type) replicons in Escherichia coli stringent and relaxed strains starved for particular amino acids; Wrobel B et al.; It was demonstrated previously that ColE1-type plasmids, the most commonly used vectors in molecular cloning, can be amplified in amino acid-starved relA mutants of Escherichia coli . Subsequent studies demonstrated that replication of at least some plasmids during amino acid starvation depends not only on the host relA allele but also on temperature and on the nature of deprived amino acid . Therefore, we investigated efficiency of amplification of two types of ColE1 plasmids (pMB1- and p15A-derived replicons) in E . coli relA+ and relA- hosts starved for different amino acids at 30 degrees C, 37 degrees C and 43 degrees C . We found differential amplification efficiency of plasmids pBR328 (pMB1-derived replicon) and pACYC184 (p15A-derived replicon) in the relA mutant during starvation for particular amino acids . Although amplification of pBR328 was negligible in the relA+ host, significant increase in pACYC184 content was observed in this strain starved for some (but not all) amino acids . The amplification efficiency of pBR328 and pACYC184 was found to be dependent on temperature . These results indicate that for maximal amplification of particular plasmid appropriate amino acid starvation and optimal temperature should be chosen . Our findings are in agreement with recently proposed model of the regulation of ColE1-type plasmid replication in amino acid-starved E . coli cells.

Mech Ageing Dev, 1997 Dec, 98(3), 189 - 202
Transgenic mouse models for studying mutations in vivo: applications in aging research; Vijg J et al.; To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed . The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations . Procedures have been worked out to efficiently recover the plasmids into E . coli host cells . A positive selection system, permitting only E . coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered . Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain . Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Dev Growth Differ, 1997 Aug, 39(4), 515 - 22
Double-segment defining role of even-skipped homologs along the evolution of insect pattern formation; Xu X et al.; Recent studies on insect patterning suggest that the genetic hierarchy may be roughly conserved in phylogenetically divergent species, but pair-rule genes may not function identically in all insects . In order to understand potential evolutionary changes in the role of the pair-rule genes, a Bombyx even-skipped homolog was cloned and its expression pattern during early embryogenesis studied . Eight stripes of Bombyx even-skipped were progressively expressed in an antero-posterior order . Later, these stripes disappeared anteriorly . Under this detection system, Bombyx even-skipped stripes clearly do not resolve into the corresponding secondary stripes, an obvious difference from Drosophila and Tribolium . These results suggest that Bombyx even-skipped may serve a double-segment defining role and may determine the odd-numbered engrailed stripes.

Biochimie, 1997 Jul, 79(7), 435 - 8
On the use of Zn2+ to discriminate endonucleases activated during apoptosis; Torriglia A et al.; One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease . Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis . Acidic DNases were thought to be insensitive to Zn2+ . In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells . These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei . We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM . At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme . This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis . Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.

Acta Trop, 1997 Oct 14, 68(1), 23 - 35
Detection of lectin activity in Leishmania promastigotes and amastigotes; Svobodova M et al.; Cell lysates from 16 strains of eight Leishmania species were used to test haemagglutination activity (HA) against a variety of RBC . HA was detected using native or neuraminidase-treated rabbit RBC; it was found in promastigotes of all the Leishmania strains tested and in axenic amastigotes of L . mexicana . The HA was trypsin-sensitive, heat-resistant and partially dependent on divalent cations . The HA was inhibited by amino-sugars, LPS from E . coli K 235, fetuin and heparin . The HA is probably located on the surface of promastigotes, as shown by the same sugar-binding specificity when live cells were used in inhibition tests . Leishmania promastigotes were agglutinated with neoglycoproteins NAc-glc-BSA and NAc-gal-BSA . This agglutination was blocked by galactosamine, glucosamine and sialic acid, but not by glcNAc or galNAc . The level of HA is increased in axenic amastigotes when compared to promastigotes . In general, HA was found at a higher titre in infective compared to uninfective strains of Leishmania . These results suggest that the haemagglutinin could play a role in the vertebrate phase of the parasite life cycle, possibly in macrophage attachment or invasion.

Drug Metab Dispos, 1997 Nov, 25(11), 1228 - 33
Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents . Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins; Schmiedlin-Ren P et al.; Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates . It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium . We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice . To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4 . We found that grapefruit oil and two furanocoumarin constituents (6', 7'-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration . The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo . Assays of various juices confirmed that 6',7'-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 microM) for loss of midazolam 1'-hydroxylase activity determined in the Caco-2 cells . Studies with recombinant CYP3A4 revealed that 6', 7'-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme . We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6',7'-dihydroxybergamottin although other furanocoumarins probably also contribute.

EMBO J, 1997 Nov 3, 16(21), 6574 - 83
DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC; Weigel C et al.; The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy . DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay . DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant . This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC . DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4 . In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e . the affinity is reduced to approximately that of DnaA box R2 . Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.

EMBO J, 1997 Nov 3, 16(21), 6548 - 58
The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites; Gorman MA et al.; The structure of the major human apurinic/ apyrimidinic endonuclease (HAP1) has been solved at 2.2 A resolution . The enzyme consists of two symmetrically related domains of similar topology and has significant structural similarity to both bovine DNase I and its Escherichia coli homologue exonuclease III (EXOIII) . A structural comparison of these enzymes reveals three loop regions specific to HAP1 and EXOIII . These loop regions apparently act in DNA abasic site (AP) recognition and cleavage since DNase I, which lacks these loops, correspondingly lacks AP site specificity . The HAP1 structure furthermore suggests a mechanism for AP site binding which involves the recognition of the deoxyribose moiety in an extrahelical conformation, rather than a 'flipped-out' base opposite the AP site.

EMBO J, 1997 Nov 3, 16(21), 6384 - 93
SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane; Matsumoto G et al.; SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation . We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction . We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein . Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex . We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo . One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein . The SecA36 protein could also insert into the mutant membrane vesicles in vitro . These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.

Structure, 1997 Oct 15, 5(10), 1373 - 83
The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology; Mao C et al.; BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate . In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates . The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E . coli PNP but not the human enzyme . The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction . Structural comparison of the active sites of mammalian and E . coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E . coli PNP . RESULTS: The crystal structure of E . coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E . coli uridine phosphorylase . Nevertheless, even though the overall geometry of the E . coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different . In E . coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar . The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues . In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP . CONCLUSIONS: The structures of human and E . coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs . This structural data may be useful in designing prodrugs that can be activated by E . coli PNP but not the human enzyme.

Structure, 1997 Oct 15, 5(10), 1261 - 4
La cage aux fold: asymmetry in the crystal structure of GroEL-GroES-(ADP)7; Harrison CJ; The structure of the molecular chaperone GroEL from Escherichia coli in complex with GroES and seven ADP molecules has recently been reported to 3 A resolution . The structure illustrates how the cavity of GroEL is converted from a hydrophobic environment, suitable for binding unfolded polypeptides, to a much larger hydrophilic environment suitable for refolding proteins.

Cell Mol Life Sci, 1997 Aug, 53(8), 681 - 8
Recombinant synthesis of mouse Zn3-beta and Zn4-alpha metallothionein 1 domains and characterization of their cadmium(II) binding capacity; Capdevila M et al.; Genetic engineering, coupled with spectroscopic analyses, has enabled the metal binding properties of the alpha and beta subunits of mouse metallothionein 1 (MT) to be characterized . A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms . The cadmium(II) binding properties of recombinant Zn4-alpha MT and Zn3-beta MT have been studied by electronic absorption and circular dichroism . The former binds Cd(II) identically to alpha fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the native protein . Titration of Zn3-beta MT with CdCl2 results in the formation of Cd3-beta MT . The addition of excess Cd(II) leads to Cd4-beta MT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd9-beta MT . The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100 . The Cd3-beta MT species is stable in the presence of this strong metal-chelating agent.

Lett Appl Microbiol, 1997 Sep, 25(3), 225 - 8
Partial deletion of transposon Tn4560 integrated into the genome of Streptomyces tendae; Engel P et al.; Polymerase chain reaction (PCR), Southern hybridization and DNA sequencing experiments were done to determine whether all of Tn4560, a Streptomyces transposon, integrated into the genomes of three nikkomycin non-producing mutants . A deletion of 279 bases occurred at one end of Tn4560 while present in the genome of one of the mutants.

FEMS Microbiol Lett, 1997 Oct 15, 155(2), 223 - 9
High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes; Flett F et al.; Many streptomycetes, including S . coelicolor A3(2), possess a potent methyl-specific restriction which can present an effective barrier to the introduction of heterologous DNA . We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S . coelicolor and S . lividans 66 using two E . coli donors: the standard, methylation proficient strain S17-1, and the methylation deficient donor, ET12567(pUB307) . We demonstrate that the methylation deficient donor can yield > 10(4)-fold more S . coelicolor exconjugants than the standard donor . In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid . The conjugation procedure is efficient enough to obtain exconjugants with 'suicide' delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.

FEMS Microbiol Lett, 1997 Oct 15, 155(2), 193 - 8
Nickel resistance in Escherichia coli V38 is dependent on the concentration used for induction; Rubikas J et al.; Strain Escherichia coli V38 resistant to 4 mM NiCl2 was isolated from the city sewage sludge . It showed low nickel accumulation by cells and nickel ion efflux . Cells were pregrown (induced) overnight in the presence of Ni2+, then the culture was kept on ice for 20-30 min and transferred to 37 degrees C for further incubation . When the Ni2+ concentration during growth was the same as during incubation, there was no noticeable accumulation of Ni2+ . When the Ni2+ concentration during incubation was higher than that used for induction, uptake of 63Ni2+ and delayed efflux were seen . The uptake and delay of both efflux and growth were directly proportional to the difference between the concentrations used for induction and incubation . Active nickel ion uptake was seen in cells taken from cultures in the delayed efflux period.

Mol Microbiol, 1997 Sep, 25(6), 1031 - 46
Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family; Roof WD et al.; slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis-trans peptidyl-prolyl isomerases (PPlases) . slyD mutations affect plaque formation by the phage phiX174 by blocking the action of the phage lysis protein E . Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid . These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins . A wide variation in the plating efficiency of phiX174 on these E(R) strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity . Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds . Finally, overexpression of slyD causes filamentation of the host . Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.

J Clin Microbiol, 1997 Nov, 35(11), 2981 - 2
Virulence patterns of Escherichia coli K1 strains associated with neonatal meningitis; Bingen E et al.; The prevalence of the ibe10 gene, of the pap, afa, and sfa adhesin-encoding operons, and of a 14.9-kb rrn-containing HindIII fragment was studied for 67 Escherichia coli neonatal meningitis strains, 58 E . coli K1 commensal strains, and 47 E . coli blood isolates from neonates without meningitis . ibe10, sfa, and the 14.9-kb HindIII fragment were observed significantly more often in the meningitis strains than in blood or commensal strains.

J Clin Microbiol, 1997 Nov, 35(11), 2958 - 63
Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes: relationship with toxic phenotypes; Blanco M et al.; Seventy-four E . coli strains isolated from piglets with diarrhea or edema disease in Spain were serotyped and examined for production of heat-labile (LT) and heat-stable (ST) enterotoxins (LT-I, LT-II, STaH, STaP, and STb) and verotoxins (VT1, VT2, and VT2v = VTe) by phenotypic (Vero cell assay and infant mouse test) and genotypic (colony hybridization and PCR) methods . In general, an excellent correlation was found between the results obtained with a PCR approach and those determined with biological assays . DNA probes used in the hybridization also showed a very good agreement with phenotypic results, with the exception of a VT1 probe that initially produced 10 false-positive reactions . The gene coding for STb (58 strains) was the most prevalent gene detected by PCR, followed by those coding for STa (46 strains), LT (19 strains), VT2v (11 strains), and VT1 (1 strain) . Apparently, in Spain three seropathotypes are predominant: (i) O149:K91:H10 K88+ LT-I+ STb+, (ii) O141:K85ab:H- P987+ STaP+, and (iii) O138:K81:H14 or H- STaP+ VT2v+ . We conclude that PCR is a fast, specific, and practical method for the identification of enterotoxin and VT genes in clinical and epidemiological studies.

J Clin Microbiol, 1997 Nov, 35(11), 2752 - 8
Heterogeneity of BmpA (P39) among European isolates of Borrelia burgdorferi sensu lato and influence of interspecies variability on serodiagnosis; Roessler D et al.; The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed . The bmpA sequences of 12 isolates representing all three species of B . burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced . The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B . burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity) . The interspecies identities ranged from 86 to 92% . Cluster analysis of BmpA reflected the subdivision of B . burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B . garinii strains . The BmpA protein of each species of B . burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies . Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B . afzelii and B . garinii . A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B . burgdorferi sensu stricto but no or only weak reactivity with BmpA of B . garinii and B . afzelii, respectively . Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100% . IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%) . With the BmpA proteins of B . afzelii and B . garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B . burgdorferi sensu stricto . Therefore, we recommend that recombinant BmpA of B . afzelii or B . garinii should be used solely, or in addition to B . burgdorferi sensu stricto BmpA, in serodiagnostic tests for Lyme borreliosis in Europe.

Biochem Mol Biol Int, 1997 Oct, 43(2), 391 - 8
Cloning and sequencing of the secY gene homolog from Mycobacterium bovis BCG; Kim JK et al.; The complete nucleotide sequence of a 1,513 bp fragment of Mycobacterium bovis BCG containing the secY gene homolog and partial adk gene that encodes an adenylate kinase has been determined . The secY gene of BCG has an open reading frame of 441 amino acids with homology to the SecY protein family . Comparative analyses of the deduced amino acid sequence of additional partial ORF revealed strong similarity to the known adenylate kinases.

J Med Microbiol, 1997 Jun, 46(6), 506 - 10
Pulsed-field gel electrophoresis genomic fingerprinting of hospital Escherichia coli bacteraemia isolates; Blackwood RA et al.; Pulsed-field gel electrophoresis (PFGE), because of the increased sensitivity it affords over other methods of bacterial genotyping, represents a potentially powerful tool for the characterisation of isolates from hospital infections . Genomic fingerprinting by PFGE was applied to all clinical isolates of Escherichia coli obtained from blood during a 6-month period (78 isolates, 58 patients) at the University of Michigan Medical Center . The rare-restriction patterns of these isolates, in contrast to those of isolates from the E . coli reference collection (ECOR), were not randomly distributed through the E . coli species . Four related clusters, which represented c . 21% of the blood isolates, were identified . Two of these genotypic clusters were also clustered temporally, their members all being isolated within the same 2-week period, while the other two clusters spanned the study period . These observations indicate in-hospital endemic vectors or the occurrence of specialised E . coli lineages that are capable of invading the bloodstream and exploiting in-hospital vectors, or both.

Mol Cell Biochem, 1997 Oct, 175(1-2), 117 - 23
Impaired phosphatidylcholine biosynthesis and ascorbic acid depletion in lung during lipopolysaccharide-induced endotoxaemia in guinea pigs; Benito E et al.; Injection of guinea pigs with a single dose of Escherichia coli lipopolysaccharide (3.2 mg/100 g) induces a reversible endotoxic shock that was evaluated by measuring plasma glucose levels and aspartate aminotransferase activity at 24 h after lipopolysaccharide injection . The hypoglycaemia and the increase in plasma aminotransferase activity observed, correlated with the alterations found during the recovery phase of endotoxic shock . When lipid peroxidation and some antioxidant systems were measured in lungs from treated animals, we only found differences in ascorbic acid content, that was decreased by 50% . Lipopolysaccharide treatment results in a depression of pulmonary phosphatidylcholine synthesis, that correlates with the surfactant deficiencies associated with respiratory illnesses in septic shock . Guinea pigs fed on a diet with a low content in ascorbic acid were more sensitive to endotoxin . In these animals we found no detectable levels of ascorbic acid in lung, whereas both vitamin E lung levels and pulmonary phosphatidylcholine synthesis were significantly decreased . Our results point out the significance of ascorbic acid in the protection against oxidative lung injury associated to endotoxaemia, and validate our shock model for further studies on the mechanisms of this pathological condition.

Genet Anal, 1997 Jul, 14(2), 47 - 50
Rapid method for detection of point mutations using mismatch binding protein (MutS) and an optical biosensor; Gotoh M et al.; This paper describes a new method for detecting DNA point mutations using a mismatch binding protein . The interactions of mismatches and mismatch binding proteins are detected by the optical biosensor technology based on surface plasmon resonance (SPR).

J Nutr, 1997 Nov, 127(11), 2253 - 9
Glutamine supplementation maintains intramuscular glutamine concentrations and normalizes lymphocyte function in infected early weaned pigs; Yoo SS et al.; Numerous studies in humans and rats have shown that glutamine supplementation during stressful conditions has favorable outcomes . However, the requirements for glutamine during weaning are unknown . Thus, the effects of glutamine supplementation in healthy and infected weaned pigs were investigated . At 21 d of age, pigs were weaned to an elemental diet supplemented with glutamine (+Gln) or an isonitrogenous diet containing nonessential amino acids (-Gln) . At 26 d of age, pigs were intraperitoneally injected with Escherichia coli (+Ecoli) or buffered saline (-Ecoli) and killed at 28 d of age . Infection decreased (P < 0.05) plasma and intramuscular glutamine concentrations, but infected pigs that received +Gln diets had higher intramuscular glutamine levels than those that received -Gln diets . Infected pigs had elevated (P < 0.05) total leukocyte counts, and blood lymphocyte responses ({3H}-thymidine incorporation) to a mixture of phorbol myristate acetate and ionomycin were reduced . White blood cell counts were greater (P < 0.05) in +Gln than -Gln pigs . The peak responses to concanavalin A (Con A) by lymphocytes of +Ecoli+Gln pigs were greater (P < 0.05) than those of +Ecoli-Gln pigs and not different than those of noninfected pigs . Hence, glutamine supplementation maintained muscular glutamine concentrations and normalized lymphocyte function in infected pigs.

J Nutr, 1997 Nov, 127(11), 2151 - 7
Low glutamine concentrations induce phenotypical and functional differentiation of U937 myelomonocytic cells; Spittler A et al.; L-Glutamine is the most abundant free amino acid of the human body and is essential for the culture of many cell types . Clinically, reduction of glutamine by administration of glutaminase or the use of glutamine analogs is a common therapy for patients with acute lymphocytic leukemia . In the current study, we investigated the influence of glutamine concentrations on the human myelomonocytic cell line U937 . Decreasing the glutamine concentration evoked a reduction in DNA synthesis (R2 = 0.9885, P < 0.0001), increased cell volume (P < 0.01) and the cytoplasm/nuclear ratio, and enhanced the development of vacuoles but did not influence cell viability . Culturing cells in reduced concentrations of glutamine augmented the percentage of cells expressing CD64 (Fc receptor for IgG/FcgammaRI, P < 0.01), CD11b (complement receptor type 3/CR3, P < 0.001) and CD71 (transferrin receptor, P < 0.05) . The percentage of U937 cells expressing CD23 (low affinity receptor for IgE/FcepsilonRII) was increased at low concentrations of glutamine at both the protein (P < 0.01) and mRNA levels . The percentage of U937 cells phagocytizing opsonized E . coli (P < 0.001) or latex particles (P < 0.001) was enhanced by lowering the glutamine concentration . In conclusion, reducing glutamine concentration causes differentiation of the cell line U937 along the monocytic pathway . These effects may indicate a mechanistic basis for prior published evidence that glutaminase and glutamine antagonists are effective anti-tumor agents.

J Exp Med, 1997 Nov 3, 186(9), 1547 - 56
Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers; Kuo FC et al.; B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes . As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion . To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site . This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase . Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues . In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs . Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Blood, 1997 Nov 1, 90(9), 3496 - 506
Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver; Humeau L et al.; Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene . Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice . Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice . Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.

J Biol Chem, 1997 Oct 10, 272(41), 26017 - 22
Covalent modification of an exposed surface turn alters the global conformation of the biotin carrier domain of Escherichia coli acetyl-CoA carboxylase; Chapman-Smith A et al.; We have studied the apo (unbiotinylated) and holo (biotinylated) forms of BCCP87, an 87-residue COOH-terminal peptide comprising the biotin carrier domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase . The apo protein spontaneously formed disulfide-linked dimers and was modified readily by sulfhydryl reagents, whereas the holo protein remained monomeric and was unreactive toward sulfhydryl reagents unless a protein denaturant was present . These data indicated that the single cysteine residue of the domain (Cys-116) was much more reactive in the apo form of the protein . Incubation of apoBCCP87 with biotin ligase for different times, followed by reaction with fluorescein-5-maleimide, clearly showed that the loss of Cys-116 reactivity was the result of modification with biotin . In addition, reaction of Cys-116 with 5,5'-dithiobis(2-nitrobenzoic acid) showed that apoBCCP87 denatured at lower urea concentrations than holoBCCP87 . We also found that apoBCCP87 was at least 10-fold more sensitive than the holo form to proteolysis by a range of proteases . Identification of the cleavage sites indicated that the differences in protease sensitivity could not be attributed to shielding of susceptible bonds by the biotin moiety of the holo protein . These data indicate that a conformational change accompanies biotinylation of the biotin domain . Thus, modification of a beta-turn protruding from the protein surface results in alteration of the overall structure of this protein domain.

J Cell Biochem, 1997 Oct 1, 67(1), 143 - 53
Transgene-coded chimeric proteins as reporters of intracellular proteolysis: starvation-induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans; Zdinak LA et al.; The product of an integrated transgene provides a convenient and cell-specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans . The transgene is an in-frame fusion of a 5'-region of the C . elegans unc-54 (muscle myosin heavy-chain) gene to the lacZ gene of Escherichia coli {Fire and Waterston (1989): EMBO J 8:3419-3428}, encoding a 146-kDa fusion polypeptide that forms active beta-galactosidase tetramers . The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h . The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre-existing proteases . Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h . Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) protein . Some of these intermediates are conjugated to ubiquitin . We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin-mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer.

Virology, 1997 Sep 29, 236(2), 338 - 47
Identification of a novel peptide substrate of HSV-1 protease using substrate phage display; O'Boyle DR 2nd et al.; The method of substrate phage display was used to select a preferred substrate from three monovalent display libraries using the HSV-1 protease . The display libraries consisted of four random amino acids, six random amino acids, and a biased library containing four amino acids from the P side of the HSV-1 maturation site followed by four random amino acids . A series of consensus peptides was synthesized based upon the results from these screens and tested in peptide cleavage assays . An eight amino acids consensus peptide (LVLASSSF) derived from the phage results was cleaved as efficiently as a 20-mer maturation site peptide . The selected amino acid sequences also allowed the design of a four amino acid paranitroanilide substrate for continuous assay of HSV-1 protease . Similar to HCMV protease, these results define P4 to P1 as a minimal substrate recognition domain for the HSV-1 protease and suggest that P4 to P1 is the minimal substrate domain which all herpesvirus proteases recognize.

Carcinogenesis, 1997 Sep, 18(9), 1785 - 91
Sensitivity of Escherichia coli (MutT) and human (MTH1) 8-oxo-dGTPases to in vitro inhibition by the carcinogenic metals, nickel(II), copper(II), cobalt(II) and cadmium(II); Porter DW et al.; The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in cultured cells, has been associated with generation of the promutagenic lesion 8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects . One possible source of this base may be 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool, from which it is incorporated into DNA . To promote such incorporation, the metals would have to inhibit specific cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool . The present study was designed to test such inhibition in vitro on 8-oxo-dGTPases from two different species, the human MTH1 protein and Escherichia coli MutT protein . In the presence of Mg(II), the natural activator of 8-oxo-dGTPases, all four metals were found to inhibit both enzymes . For MTH1, the IC50 values (+/- SE; n = 3-4) were 17 +/- 2 microM for Cu(II), 30 +/- 8 microM for Cd(II), 376 +/- 71 microM for Co(II) and 801 +/- 97 microM for Ni(II) . For MutT, they were 60 +/- 6 microM for Cd(II), 102 +/- 8 microM for Cu(II), 1461 +/- 96 microM for Ni(II) and 8788 +/- 1003 microM for Co(II) . Thus, Cu(II) and Cd(II) emerged as much stronger inhibitors than Ni(II) and Co(II), and MTH1 appeared to be generally more sensitive to metal inhibition than MutT . Interestingly, in the absence of Mg(II), the activity of the enzymes could be restored by Co(II) to 73% of that with Mg(II) alone for MutT, and 34% for MTH1, the other metals being much less or non-effective . The difference in sensitivity to metal inhibition between the two enzymes may reflect the differences in the amino acid ligands, especially the cysteine ligand, outside their evolutionarily conserved Mg(II)-binding active sites, which might indicate predominantly non-competitive or uncompetitive mechanism of the inhibition . The overall results suggest that inhibition of 8-oxo-dGTPases may be involved in the mechanisms of induction of the 8-oxoguanine lesion in DNA by the metal ions studied, especially the non-redox-active Cd(II) cation.

Bioconjug Chem, 1997 Sep-Oct, 8(5), 695 - 701
Genetic construction and characterization of an anti-monkey CD3 single-chain immunotoxin with a truncated diphtheria toxin; Ma S et al.; We have previously developed a chemically conjugated anti-rhesus monkey CD3 immunotoxin FN18-CRM9 that can deplete in vivo T cells and induce long term tolerance of mismatched renal allograft in rhesus monkeys . This immunotoxin is a monkey analogue of anti-human CD3 immunotoxin UCHT1-CRM9 . In this study, we cloned the light and heavy chain variable regions of anti-monkey CD3 monoclonal antibody FN18 and constructed a single-chain Fv (sFv) by linking variable light and variable heavy regions with a (Gly4Ser)3 linker . The single-chain immunotoxin DT390-FN18sFv was constructed by ligating the sFv to the carboxyl terminus of DT390, a truncated form of diphtheria toxin . The DT390-FN18sFv fusion protein was expressed in Escherichia coli and purified with Ni-RTA affinity and anion exchange columns . Similar to the chemically conjugated immunotoxin FN18-CRM9, DT390-FN18sFv can also specifically inhibit protein synthesis in primary monkey T cells in a dose-dependent manner . DT390-FN18sFv at 10(-7) mol/L or FN18-CRM9 at 10(-8) mol/L is sufficient to reduce protein synthesis of monkey primary T cells to less than 5% of the control . The 50% inhibition dosage (IC50) of FN18-CRM9 is 1 x 10(-10) mol/L, while the IC50 of DT390-FN18sFv is 1 x 10(-8) mol/ L, reflecting the lowered affinity of monovalent Fab' FN18 to its parental divalent antibody . The availability of functional FN18sFv will provide the basis for the construction of divalent anti-CD3 immunotoxins for preclinical studies on the induction of tolerance in organ transplantation and experimental autoimmune diseases.

Mol Gen Genet, 1997 Aug, 255(6), 605 - 10
Analysis of the cis-acting DNA elements required for piggyBac transposable element excision; Elick TA et al.; The terminal DNA sequence requirements for piggyBac transposable element excision were explored using a plasmid-based assay in transfected, cultured insect cells . A donor plasmid containing duplicate 3' piggyBac terminal inverted repeats was constructed that allowed individual nucleotides or groups of nucleotides within one of the 3' repeats to be mutated . The relative extent of excision using the mutated end versus the wild-type end was then assayed . Removal of even one of the terminal 3' G nucleotides from the piggyBac inverted repeat, or removal of the dinucleotide AA from the flanking TTAA target site prevents excision of piggyBac at the mutated terminus . Incorporation of an asymmetric TTAC target site at the 3' end does not prevent excision from the mutated end . Thus, both piggyBac DNA and flanking host DNA appear to play crucial roles in the excision process.

J Hered, 1997 Sep-Oct, 88(5), 380 - 3
The use of a wild pig x domestic pig intercross to map phenotypic trait loci; Andersson L; Intercrosses between divergent lines of domestic animals adapted to different environmental conditions and/or production systems provide unique opportunities for mapping phenotypic trait loci . The strategy resembles the use of interspecific crosses developed for general linkage mapping . We have made an intercross between the wild pig and domestic pigs of the Large White breed . A comprehensive linkage map comprising more than 230 genetic markers has been established . The successful use of this intercross for mapping loci for some monogenic traits (coat color and an intestinal receptor for E . coli K88 pili) as well as quantitative trait loci (QTLs) with major effects of growth and fatness is reviewed.

Biochemistry, 1997 Oct 14, 36(41), 12395 - 9
Nickel inhibits binding of alpha2-macroglobulin-methylamine to the low-density lipoprotein receptor-related protein/alpha2-macroglobulin receptor but not the alpha2-macroglobulin signaling receptor; Odom AR et al.; A previous study demonstrated that activated alpha2-macroglobulin (alpha2M*) binding to the low-density receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) is blocked by Ni2+ {Hussain, M . M., et al . (1995) Biochemistry 34, 16074-16081} . We now report that the effect of Ni2+ is on a region of the alpha2M molecule upstream of the carboxyl terminal receptor recognition domain . This observation is consistent with previous observations from this laboratory suggesting that alpha2M* binding to LRP/alpha2MR involves a region of the alpha2M molecule immediately upstream of the receptor recognition domain {Enghild, J . J., et al . (1989) Biochemistry 28, 1406-1412} . We further demonstrate that Ni2+ has no effect on the binding of alpha2M* or a cloned and expressed receptor binding fragment (RBF) to the recently described alpha2M signaling receptor as assessed by direct binding and signal transduction studies.

Biol Chem, 1997 Sep, 378(9), 975 - 82
The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB; Gast FU et al.; McrBC is a GTP-dependent restriction endonuclease of E . coli K12, selectively directed against DNA containing modified cytosine residues . McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-Pu(m)C sites separated by 40-80 base pairs . Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-Pu(m)C sites in DNA can be sufficient to elicite binding by McrB . Binding to such substrates is, however, weak and strongly dependent on the sequence context of Pu(m)C sites . (ii) Strong DNA binding (K(ass) approximately 10(7)M{-1}) is dependent on the presence of at least two Pu(m)C sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT- --> -A(m)CT(C/G)AGT- --> -AGG(m)CCT- --> -AAG(m)CTT-) . (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP . (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates . (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding . (vi) Mutations in McrB which lead to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.

J Biochem (Tokyo), 1997 Sep, 122(3), 627 - 34
Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase; Asano Y et al.; Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA . We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit . Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease . The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease . The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and Pllb starting from residue 298; and the third fragment, P11c, starting from residue 458 . Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i.e., the amino terminal-proximal site I located at the boundary between P6 and Pllb, and the carboxy terminal proximal site II on P11c fragment . The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases . Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively . From the V8 concentration dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains . The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products.

J Med Assoc Thai, 1997 Sep, 80 Suppl 1, S129 - 37
Production and evaluation of Taq DNA polymerase; Leelayuwat C et al.; Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories . In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E . coli . The enzyme was characterized and evaluated in comparison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A . The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protein with the total of 448,000 units/litre of the bacterial culture . The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis . Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus . Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations . In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR . The cost of enzyme preparation was about 256 times less than that of the commercial enzyme . Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.

J Biol Chem, 1997 Oct 31, 272(44), 28066 - 72
Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana . Structural and functional relationship to yeast old yellow enzyme; Schaller F et al.; Using partial amino acid sequence information for 12-oxophytodienoate-10,11-reductase obtained from Corydalis sempervirens we have cloned the homologous enzyme from Arabidopsis thaliana . The open reading frame of the cDNA encodes a polypeptide of 372 amino acids (Mr = 41,165) with significant similarity to the sequence of Old Yellow Enzyme from Saccharomyces carlsbergensis (Saito, K., Thiele, D . J., Davio, M., Lockridge, O., and Massey, V . (1991) J . Biol . Chem . 266, 20720-20724), a flavin (FMN)-protein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyls . Specifically, all residues required for binding of FMN in Old Yellow Enzyme are conserved in the A . thaliana sequence, as are all residues associated with catalytic activity . The enzyme was functionally expressed from its cDNA in Escherichia coli and thus proven to encode OPDA reductase . Further similarities of OPDA reductase and yeast Old Yellow Enzyme include their binding to and elution by reductant from N-(4-hydroxybenzoyl)aminohexyl-Sepharose, the immunoreactivity of yeast Old Yellow Enzyme with an antiserum raised against plant OPDA reductase and the demonstration that Old Yellow Enzyme is an active OPDA reductase . It is thus conceivable that the physiological role of Old Yellow Enzymes now known from bacteria, yeasts, and higher plants, is in oxylipin metabolism.

J Biol Chem, 1997 Oct 31, 272(44), 27994 - 8000
Relative functions of the alpha and beta subunits of the proteasome activator, PA28; Song X et al.; PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome . PA28 is composed of two homologous subunits, alpha and beta, arranged in alternating positions in a ring-shaped oligomer with a likely stoichiometry of (alphabeta)3 . Our previous work demonstrated that the carboxyl terminus of the alpha subunit was necessary for PA28 to bind to and activate the proteasome . The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the alpha and beta subunits in proteasome activation . Each subunit and various mutants of the alpha subunit were expressed in Escherichia coli and purified . PA28alpha stimulated the proteasome, but had a much greater Kact than native heteromeric PA28 . In contrast, PA28beta was unable to stimulate the proteasome . Mutants of the alpha subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome . However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents . Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28alpha . Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28alpha, had no effect on proteasome stimulatory activity . Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits . PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein . PA28 molecules reconstituted from inactive alpha subunits and wild type beta subunits remained inactive . However, PA28 molecules reconstituted from suboptimally active alpha mutants and wild type beta subunits had the same activity as native heteromeric PA28 . These results indicate that the beta subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.

J Biol Chem, 1997 Oct 31, 272(44), 27919 - 30
Fidelity of Escherichia coli DNA polymerase III holoenzyme . The effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies; Bloom LB et al.; The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay . Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies finc = 5.6 x 10(-6), 4.2 x 10(-7), and 7 x 10(-7), respectively . Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 10(-4), attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G . At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide . As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide . Compared with pol III holoenzyme, the alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs . Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha subunit . An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D . K., and Goodman, M . F . (1997) J . Biol . Chem . 272, 27931-27935 (accompanying paper)).

J Biol Chem, 1997 Oct 31, 272(44), 27707 - 15
Local folding of the N-terminal domain of Escherichia coli RecA controls protein-protein interaction; Masui R et al.; To obtain structural information about the self-association of the protein RecA, we studied urea denaturation of RecA by circular dichroism spectroscopy and gel filtration . Gel filtration analysis showed that urea at low concentrations, 1.0-1.2 M, dissociated the RecA oligomer to almost a monomeric state prior to the unfolding of each molecule . Upon treatment with 1.0 M urea, the circular dichroism spectrum showed a decrease in the alpha-helical content of RecA . A similar decrease was observed in the absence of urea for RecA at an extremely low protein concentration; the RecA oligomer dissociated to an almost completely monomeric state . The properties of RecA at low urea concentrations were similar to those of a truncated RecA lacking the first 33 N-terminal residues (Delta33RecA) . Addition of a synthetic peptide corresponding to the 33 N-terminal residues to Delta33RecA increased the alpha-helical content . These results suggest that local folding of the N-terminal domain is coupled to protein-protein interactions of monomeric RecA, which are involved in the regulation of filament formation . The dissociation constant for interaction between RecA monomers was determined from the ellipticity data to be 0.1 microM.

J Biol Chem, 1997 Oct 31, 272(44), 27652 - 9
Inactivation of dehydratase {4Fe-4S} clusters and disruption of iron homeostasis upon cell exposure to peroxynitrite; Keyer K et al.; Phagocytes produce both nitric oxide and superoxide as components of the oxidative defense against pathogens . Neither molecule is likely at physiological concentrations to kill cells . However, two of their reaction products, hydrogen peroxide and peroxynitrite, are strong oxidants, cell-permeant, and toxic . Hydrogen peroxide generates oxidative DNA damage, while the primary mechanism of toxicity of peroxynitrite has not yet been determined . Recent in vitro studies indicated that peroxynitrite is capable of oxidizing the {4Fe-4S} clusters of a family of dehydratases (Hausladen, A., and Fridovich, I . (1994) J . Biol . Chem . 269, 29405-29408; Castro, L., Rodriguez, M., and Radi, R . (1994) J . Biol . Chem . 269, 29409-29415) . We demonstrate here that peroxynitrite at 1% of its lethal dose almost fully inactivated the labile dehydratases in Escherichia coli . The rate at which peroxynitrite inactivated the clusters substantially exceeded the rate at which it oxidized thiols or spontaneously decomposed . These results suggest that these dehydratases may be primary targets of peroxynitrite in vivo . Another consequence of the cluster damage was the release of 100 microM iron into the cytosol . During phagocytosis, this intracellular free iron could increase lethal DNA damage by hydrogen peroxide or protein modification by additional peroxynitrite . In response to peroxynitrite challenges, E . coli rapidly sequestered the intracellular free iron using an undefined scavenging system . The iron-sulfur clusters were more gradually repaired by a process that drew iron from its iron-storage proteins . These are likely to be critical events in the struggle between phagocyte and pathogen.

J Biol Chem, 1997 Oct 31, 272(44), 27589 - 97
Glycogenin-2, a novel self-glucosylating protein involved in liver glycogen biosynthesis; Mu J et al.; Glycogenin is a self-glucosylating protein involved in the initiation phase of glycogen biosynthesis . A single mammalian gene had been reported to account for glycogen biogenesis in liver and muscle, the two major repositories of glycogen . We describe the characterization of novel forms of glycogenin, designated glycogenin-2 (GN-2), encoded by a second gene that is expressed preferentially in certain tissues, including liver, heart, and pancreas . Cloning of cDNAs encoding glycogenin-2 indicated the existence of multiple species, including three liver forms (GN-2alpha, GN-2beta, and GN-2gamma) generated in part by alternative splicing . Overall, GN-2 has 40-45% identity to muscle glycogenin but is 72% identical over a 200-residue segment thought to contain the catalytic domain . GN-2 expressed in Escherichia coli or COS cells is active in self-glucosylation assays, and self-glucosylated GN-2 can be elongated by skeletal muscle glycogen synthase . Antibodies raised against GN-2 produced in E . coli recognized proteins of Mr approximately 66,000 present in extracts of rat liver and in cultured H4IIEC3 hepatoma cells . In H4IIEC3 cells, most of the GN-2 was present as a free protein but some was covalently associated with glycogen fractions and was only released by treatment with alpha-amylase . H4IIEC3 cells also expressed the muscle form of glycogenin (glycogenin-1), which was attached to a chromatographically separable glycogen fraction.

J Biol Chem, 1997 Oct 31, 272(44), 27572 - 6
A mutant truncated protein disulfide isomerase with no chaperone activity; Dai Y et al.; A mutant human protein disulfide isomerase with the COOH-terminal 51 amino acid residues deleted (abb'a') has been expressed in Escherichia coli . Its secondary structures are very similar to those of the native bovine enzyme . The mutant enzyme shows neither peptide binding ability nor chaperone activity in assisting the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase but keeps most of the catalytic activities for reduction of insulin and isomerization of scrambled ribonuclease . It assists the reactivation of denatured and reduced proteins containing disulfide bonds, acid phospholipase A2, and lysozyme to different levels, which are significantly lower than those by the native bovine enzyme.

J Biol Chem, 1997 Oct 31, 272(44), 27517 - 20
pH-dependent stability and conformation of the recombinant human prion protein PrP(90-231); Swietnicki W et al.; A recombinant protein corresponding to the human prion protein domain encompassing residues 90-231 (huPrP(90-231)) was expressed in Escherichia coli in a soluble form and purified to homogeneity . Spectroscopic data indicate that the conformational properties and the folding pathway of huPrP(90-231) are strongly pH-dependent . Acidic pH induces a dramatic increase in the exposure of hydrophobic patches on the surface of the protein . At pH between 7 and 5, the unfolding of hPrP(90-231) in guanidine hydrochloride occurs as a two-state transition . This contrasts with the unfolding curves at lower pH values, which indicate a three-state transition, with the presence of a stable protein folding intermediate . While the secondary structure of the native huPrP(90-231) is largely alpha-helical, the stable intermediate is rich in beta-sheet structure . These findings have important implications for understanding the initial events on the pathway toward the conversion of the normal into the pathological forms of prion protein.

J Biol Chem, 1997 Oct 31, 272(44), 27513 - 6
Cleavage of single strand RNA adjacent to RNA-DNA duplex regions by Escherichia coli RNase H1; Lima WF et al.; RNase H1 from Escherichia coli cleaves single strand RNA extending 3' from an RNA-DNA duplex . Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9-17 nucleotides were designed to create overhanging single strand RNA regions extending 5' and 3' from the RNA-DNA duplex . Digestion of single strand RNA was observed exclusively within the 3' overhang region and not the 5' overhang region . RNase H digestion of the 3' overhang region resulted in digestion products with 5'-phosphate and 3'-hydroxyl termini . The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex . RNase H digestion of the 3' overhang region was not observed for a substrate that contained a 2'-methoxy antisense strand . The introduction of 3 deoxynucleotides at the 5' terminus of the 2'-methoxy antisense oligonucleotide resulted in cleavage . These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.

Biomed Pept Proteins Nucleic Acids, 1994-95, 1(1), 39 - 44
Chemical synthesis and characterisation of rat chaperonin 10: effect of chain length, ions, heat and N-terminal acetylation on unchaperoned folding into its heptameric form; Ball HL et al.; Recently, the sequence of mitochondrial chaperonin 10 from Rattus norvegicus (rat cpn10), with N-terminal acetylation, has been published . Two syntheses of rat cpn10 were performed, the first using a classical carbodiimide-mediated double coupling protocol (Method A) and the second a more efficient HBTU/HOBT/single coupling procedure (Method B) . The latter also involved the application of a capping procedure, using N-(2-chlorobenzyloxycarbonyloxy)succinimide {Z(2-Cl)-OSu} . The crude protein from Method A was purified using a two-step isoelectric focusing/RP-HPLC scheme and found to contain a high proportion of a deletion peptide (less Gln60) . Conversely, rat cpn10 from Method B was purified to homogeneity by one-step RP-HPLC, using a reversible lipophilic chromatographic probe . The proportion of biologically active heptameric structure was directly related to the purity of the protein and attained 84% with material from Method B . The addition of Ca/Mg ions, pH 7.2, or a heating/cooling cycle increased the proportion of heptamer for less pure protein . Shorter sequences were found not to fold into heptamers, suggesting that aggregation/folding motifs are located in 1-25 and 77-101 regions of rat cpn10 . The heptameric cpn10 (Method B) bound correctly to GroEL from E . coli, demonstrating that N-terminal acetylation is not necessary for its folding and binding to bacterial cpn60.

Biomed Pept Proteins Nucleic Acids, 1995, 1(2), 61 - 8
Heterologous expression, purification, activity and conformational studies of different forms of dianthin 30; Legname G et al.; Dianthin 30, a ribosome inactivating protein (RIP) from Dianthus caryophyllus, has been expressed in Escherichia coli . Heterologous expression of a deletion mutant dianthin 30 delta 255-270 resulted in the production of a protein identical to carnation mature dianthin 30, including the absence at the carboxy-terminal of a putative 16 amino acid long pro-signal peptide . The production of a form of dianthin 30, which includes the pro-signal, is described as well . Both dianthin 30 delta 255-270 and dianthin 30 expressed in E . coli are mainly localized (90%) in the soluble fraction . Dianthin 30 delta 255-270 and dianthin 30 have been purified to homogeneity and were shown to inhibit protein synthesis in vitro with an IC50 of 8 and of 11 ng/ml, respectively . Secondary structure analysis, carried out by circular dichroism spectroscopy, indicated that the naturally occurring and the recombinant forms of dianthin 30 and dianthin 30 delta 255-270 possess the same secondary structure composition, accounting for an alpha + beta type architecture . RIPs as immunotoxins in clinical trial and as mitotoxins in experimental models have been extremely efficacious . In addition, growing evidence indicates their effective use as antiviral agents, including in HIV-1 infection . These data indicate the ability to produce either chemically linked or recombinant fusion proteins with dianthin 30 and cell-binding ligands for production of new reagents for clinical and experimental use.

Science, 1997 Oct 31, 278(5339), 853 - 6
Metal ion chaperone function of the soluble Cu(I) receptor Atx1; Pufahl RA et al.; Reactive and potentially toxic cofactors such as copper ions are imported into eukaryotic cells and incorporated into target proteins by unknown mechanisms . Atx1, a prototypical copper chaperone protein from yeast, has now been shown to act as a soluble cytoplasmic copper(I) receptor that can adopt either a two- or three-coordinate metal center in the active site . Atx1 also associated directly with the Atx1-like cytosolic domains of Ccc2, a vesicular protein defined in genetic studies as a member of the copper-trafficking pathway . The unusual structure and dynamics of Atx1 suggest a copper exchange function for this protein and related domains in the Menkes and Wilson disease proteins.

Eur J Pharmacol, 1997 Sep 3, 334(1), 115 - 26
Probing human beta1- and beta2 -adrenoceptors with domain-specific fusion protein antibodies; Jahns R et al.; In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta1- or beta2-adrenoceptors with bacterial glutathione-S-transferase in E . coli . Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta1- or beta2-adrenoceptors in a subtype- and domain-specific manner . Antibodies directed against the second extracellular loop of the beta1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-{5,7-3H}benzimidazol-2-one ({3H}CGP 12 177), indicating a specific interaction with the native receptor . In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of {3H}CGP 12 177 . Affinity purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy . Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying beta-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

Eur J Pharmacol, 1997 Sep 3, 334(1), 99 - 102
Actions of isoform-selective and non-selective nitric oxide synthase inhibitors on endotoxin-induced vascular leakage in rat colon; Laszlo F et al.; The effects of the nitric oxide (NO) synthase inhibitor, N-(3-(aminomethyl)benzyl)-acetamidine (1400W) which is selective for the inducible isoform of NO synthase, on rat colonic microvascular injury provoked by Escherichia coli endotoxin (3 mg/kg i.v.) has been compared to those of aminoguanidine (25-50 mg/kg, s.c.), NG-iminoethyl-L-ornithine (L-NIO, 15-30 mg/kg, s.c.) and NG-nitro-L-arginine methyl ester (L-NAME, 2-5 mg/kg, s.c.) . Administration of aminoguanidine, L-NIO or L-NAME concurrently with endotoxin provoked microvascular albumin leakage 1 h later, presumably by inhibiting constitutive NO synthase, whereas 1400W (0.1-10 mg/kg, s.c.) had no such effect . Administration of all these agents during the expression of inducible NO synthase (i.e . 3 h after endotoxin challenge) attenuated the subsequent endotoxin-provoked albumin leakage 1 h later . Moreover, concurrent administration of 1400W (0.2-5 mg/kg, s.c.; doses that did not affect systemic arterial blood pressure) with endotoxin suppressed the subsequent rise in albumin leakage after 5 h . These findings indicate that 1400W is a potent inhibitor of colonic microvascular injury associated with induction of NO synthase in vivo . 1400W will thus be useful to investigate in vivo the therapeutic potential of a selective inducible NO synthase inhibitor in inflammation.

Eur J Pharmacol, 1997 Sep 3, 334(1), 67 - 73
Effects of neuropeptide FF on intestinal motility and temperature changes induced by endotoxin and platelet-activating factor; Million M et al.; Several effects of bacterial endotoxins involve an opioid pathway and neuropeptide FF is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system . The aim of this study was to investigate in rats the role of central neuropeptide FF receptors in intestinal motor disturbances and body temperature changes induced by endotoxins and platelet-activating factor (PAF), a major endotoxin mediator . Rats were fitted with intestinal electrodes, an intraperitoneal thermistor probe and an intracerebroventricular (i.c.v.) cannula for long-term use . E . coli endotoxin (100 microg/kg, i.v.) disrupted the cyclic pattern of intestinal migrating myoelectric complexes and induced a biphasic increase in body temperature while PAF (25 microg/kg, i.p.) disrupted the migrating myoelectric complexes and induced hypothermia for about 2 h . The neuropeptide FF analog, (1 DME)Y8Fa (D-Tyr-D-Leu{N-Me}-Phe-Gln-Pro-Gln-Arg-Phe-NH2) administered i.c.v . 40 and 100 microg/kg reduced the duration of migrating myoelectric complex disruption induced by endotoxin and PAF and abolished the PAF-induced hypothermia . Only at the dose of 100 microg/kg did (1 DME)Y8Fa change the biphasic endotoxin-induced hyperthermia into a monophasic increase . Naloxone (1 mg/kg, s.c.) reduced only the duration of migrating myoelectric complex disruption induced by endotoxin . These results indicate that central neuropeptide FF modulates the intestinal motor disturbances and changes in body temperature induced by endotoxin and PAF . Its action against endotoxin may involve an anti-opioid pathway whereas its action against PAF does not.

Eur J Biochem, 1997 Sep 1, 248(2), 516 - 20
Cloning and expression of the fadH gene and characterization of the gene product 2,4-dienoyl coenzyme A reductase from Escherichia coli; He XY et al.; The fadH gene coding for an NADPH-dependent 2.4-dienoyl-CoA reductase from Escherichia coli has been cloned by the polymerase chain reaction . This gene is located at 67.65 min on the E . coli chromosome . The complete open reading frame contains 2019 bp coding for the processed protein of 671 amino acid residues, with a calculated molecular mass of 72.55 kDa, which lacks the N-terminal methionine . Construction and expression of the plasmid pNDH, which contained the fadH gene under the control of the T7 promoter, resulted in a 110-fold increase in the reductase activity above the level detected in E . coli cells containing the control vector . The kinetic parameters of the purified reductase were determined to be 50 microM and 2.3 microM for the Km values of NADPH and 2-trans, 4-trans-decadienoyl-CoA, respectively, and 16 s(-1) for the k(cat) value . Analysis of the kinetic data revealed that the reaction catalyzed by this enzyme proceeds via a ping-pong mechanism . The observed dissimilarity between the E . coli and mammalian 2,4-dienoyl-CoA reductase sequences suggests that they have evolved from distinct ancestral genes . Sequence analysis also suggests that the N-terminal part of the E . coli reductase contains the FAD-binding domain whereas the NADPH-binding domain is located in the C-terminal region of the protein.

Eur J Biochem, 1997 Sep 1, 248(2), 304 - 12
Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization; Becker C et al.; Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination . This enzyme was purified from cotyledons of vetch seedlings . On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa . Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds . cDNA clones encoding proteinase A precursor have been obtained by PCR . The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence . Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence . The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings . Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6 . No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins . By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded . The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

FEMS Microbiol Lett, 1997 Oct 1, 155(1), 115 - 9
Identification and characterization of a Treponema pallidum subsp . pallidum gene encoding a DNA adenine methyltransferase; Stamm LV et al.; The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined . Southern blot analysis of T . pallidum chromosomal DNA indicated that this gene is present as a single copy . The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N6-adenine) methyltransferases . T . pallidum Dam can be assigned to group alpha DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein . Digests of T . pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam+ phenotype).

FEMS Microbiol Lett, 1997 Oct 1, 155(1), 63 - 6
Evidence of rare codon clusters within Escherichia coli coding regions; Phoenix DA et al.; It is known that there is a high occurrence of rare codons at the start of coding region . Here it is shown that although the remainder of the gene is likely to contain a relatively low number of rare codons, rare and non-rare codons do not form a random sequence . It is apparent that throughout the coding region there is a higher than expected number of rare codon clusters . For example once a rare codon has occurred there is a greater chance than expected of the next six codons containing another rare codon . This non-random distribution implies that rare codons may have an as yet unidentified biological role.

FEMS Microbiol Lett, 1997 Oct 1, 155(1), 39 - 44
Construction of a double hha hns mutant of Escherichia coli: effect on DNA supercoiling and alpha-haemolysin production; Nieto JM et al.; A double hha hns Escherichia coli mutant was constructed . The effect of the single hns mutation and of the double hha hns mutation on the expression of the alpha-haemolysin determinant of plasmid pANN202-312 was assessed . Whereas the hns mutant moderately increased expression of the toxin, the double hha hns mutant strongly enhanced transcription of the hly operon and hence expression of the toxin . This suggests that both Hha and H-NS proteins participate in the modulation of the expression of the toxin . The enhancement of haemolysin expression in the double mutant could not be correlated to a global alteration of DNA topology: DNA preparations of a reporter plasmid isolated from this mutant gave a topoisomer distribution similar to that of the parental strain.

FEMS Microbiol Lett, 1997 Oct 1, 155(1), 23 - 30
Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction; Mobasheri H et al.; Brucella usually carry two highly homologous genes (omp2a and omp2b) for porin-like proteins . In several B . abortus biovars the omp2a gene has a large deletion compared to other Brucella omp2's . In this study we have measured Omp2 pore activity in planar bilayers . Omp2b exhibits well-defined trimeric channel activity whilst Omp2a forms monomeric pores of variable size which are smaller than Omp2b . No sequence homology exists between Omp2 and porins of known structure, so hydrophobic moment analysis has been used to model their membrane topology . From this it appears likely that the deletion removes the crucial L3 internal loop.

Vet Res Commun, 1997 Oct, 21(7), 477 - 82
Failure of lipopolysaccharides to directly trigger the chemiluminescence response of isolated equine polymorphonuclear leukocytes; Benbarek H et al.; Divergent results have been reported on the effects of lipopolysaccharides (LPS) on the activation of equine polymorphonuclear leukocytes (PMN) . We therefore attempted to determine whether LPS alone can stimulate equine PMN or whether plasma factors are necessary . PMN were isolated from citrated blood on a discontinuous density gradient of Percoll . The luminol (10(-3) mol/L)-enhanced chemiluminescence (CL) of 1.25 x 10(6) cells was measured after addition of Escherichia coli LPS (0.001-10 micrograms/ml) alone or after incubation in autologous plasma (1 h, 37 degrees C) . After direct stimulation with LPS, there were random variations of CL in 16 horses that were not reproducible from one sample to the next for the same horse . LPS which had been incubated in plasma gave a dose-dependent stimulation of the CL of the PMN, which did not occur if the plasma had been heat inactivated (1 h, 56 degrees C) . These results indicated a role for plasma factors, which were unlikely to be cytokines, as there were no monocytes or lymphocytes in the plasma incubated with LPS, but might have been complement fragments or LPS ligands, such as LPS binding protein . Studies using specific antibodies against these factors are needed to clarify this question.

Mult Scler, 1996 Oct, 2(3), 125 - 32
Transcription of myelin basic protein promoted by regulatory elements in the proximal 5' sequence requires myelinogenesis; Stankoff B et al.; Myelination in the central nervous system requires synthesis by oligodendrocytes of enormous amounts of lipids and proteins for incorporation in the developing myelin membranes . To approach the regulatory events coordinating the transcriptional activation of the genes that encode myelin proteins, we examined control of the myelin basic protein (MBP) locus . MBP plays a major role in myelin compaction . During development, MBP is already expressed in mature non-myelinating oligodendrocytes . Here we show that, in transgenic animals in which the E . coli lacZ reporter gene is under the control of increasingly large portions (256, 1900 and 3200 bp) of the MBP promoter, 5' of the initiation of transcription site, reporter gene expression was initiated after myelin formation had started . This delayed expression of the transgene compared to MBP, strongly suggests that premyelinating expression is dependent on regulatory elements located outside of the 3200 bp sequence studied, while expression occurring at the time of myelin formation is dependent on the proximal promoter sequence.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 197 - 204
Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4; Twardosz A et al.; Birch pollen belongs to the most potent elicitors of Type I allergic reactions in early spring . Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated from a birch pollen expression cDNA library constructed in phage lambda gt11 . Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designated Bet v 4, with significant end to end sequence homology to EF-hand calcium-binding allergens from weed and grass pollen . Recombinant Bet v 4, expressed as beta-galactosidase fusion protein, reacted with serum IgE from approximately 20% of pollen allergic individuals . Depletion of allergenbound calcium by EGTA treatment lead to a substantial reduction of IgE-binding to Bet v 4, indicating that protein-bound calcium is necessary for the maintenance of IgE-epitopes . The greatly reduced IgE-binding capacity of clone 13, a Bet v 4 fragment that lacked the 16 N-terminal amino acids, indicated that the N-terminus contributes significantly to the proteins IgE-binding capacity . By IgE-inhibition experiments it was demonstrated that recombinant Bet v 4 shared IgE-epitopes with natural Bet v 4 and a homologous timothy grass pollen allergen . Recombinant Bet v 4 may therefore be considered as a relevant crossreactive plant allergen, which may be used for diagnosis and treatment of patients suffering from multivalent plant allergies.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 46 - 50
Stationary phase-specific mRNAs in Escherichia coli are polyadenylated; Cao GJ et al.; Polyadenylation of Escherichia coli specific mRNAs has so far been studied primarily during the exponential phase of growth . As part of an investigation of the polyadenylation of E . coli mRNAs in different physiological contexts, we studied mRNA polyadenylation in stationary phase by preparing a cDNA library from stationary phase RNA using oligodeoxythymidylate primers and analyzing the nucleotide sequence of cDNA clones corresponding to the stationary phase-specific genes, rpoS, bo1A, and dps . The sites of polyadenylation were found to be primarily in the 3'-untranslated region, either at the putative rho-independent transcription termination site (dps) or at several different sites upstream of the putative rho-independent terminator . A few examples of polyadenylation within the coding regions were also found, suggesting that nucleolytic degradation often preceded polyadenylation . In contrast to the poly(A) tracts characteristic of exponentially growing cells, many of the uncoded poly(A) tracts associated with stationary phase mRNA were interspersed with other nucleotide residues . The observation of post-transcriptional polyadenylation of specific stationary phase mRNAs in E . coli, some of which are transcribed by the RNA polymerase associated with sigma, demonstrates that mRNA polyadenylation is not confined to the exponential phase of growth.

Virology, 1997 Oct 13, 237(1), 129 - 36
Intact eukaryotic initiation factor 4G is required for hepatitis A virus internal initiation of translation; Borman AM et al.; The requirements for optimal activity of the hepatitis A virus (HAV) internal ribosome entry segment (IRES) differ substantially from those of other picornavirus IRESes . One such difference is that, to date, the HAV IRES is the only one whose efficiency is severely inhibited in the presence of the picornaviral 2A proteinase . Here we describe experiments designed to dissect the mechanism of proteinase-mediated inhibition of HAV translation . Using dicistronic mRNAs translated in vitro, we show that the HAV IRES is inhibited by the foot-and-mouth disease virus Lb proteinase, as well as by the human rhinovirus 2A proteinase . Furthermore, using mutant Lb proteinase, we demonstrate that proteolytic activity is required for inhibition of HAV IRES activity . Translation inhibition correlated closely with the extent of cleavage of the one identified common cellular target for the 2A and Lb proteinases, eukaryotic initiation factor (eIF) 4G, a component of the eIF4F cap-binding protein complex . Total rescue of HAV IRES activity was possible if purified eIF4F was added to translation extracts . In contrast, if the added eIF4F contained cleaved eIF4G, no rescue of HAV IRES activity was evidenced . Thus the HAV IRES requires intact eIF4G for activity . This is unique among the picornavirus IRESes studied to date and may help explain why HAV does not inhibit host cell translation during viral infection .

Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 552 - 6
High pressure modulation of Escherichia coli DNA gyrase activity; Chilukuri LN et al.; Elevated pressures greater than 551 bar were found to inhibit the DNA supercoiling activity of Escherichia coli DNA gyrase . A large fraction of the inhibitory effect could be reproduced by preincubation of the enzyme at high pressure prior to enzymatic assay at 1 bar . It is proposed that elevated pressure influences gyrase structure, most likely by promoting the dissociation of its subunits, however, it is also possible that effects on enzyme activity exist .

J Mol Biol, 1997 Oct 24, 273(2), 389 - 401
Release factor RF3 abolishes competition between release factor RF1 and ribosome recycling factor (RRF) for a ribosome binding site; Pavlov MY et al.; The dependence of the rate of ribosomal recycling (from initiation via protein elongation and termination, and then back to initiation) on the concentrations of release factor RF1 and the ribosome recycling factor (RRF) has been studied in vitro . High RF1 concentration was found to reduce the rate of ribosomal recycling and the extent of this reduction depended on stop codon context . The inhibitory effect of high RF1 concentrations can be reversed by a corresponding increase in RRF concentration . This indicates that RF1 and RRF have mutually exclusive and perhaps overlapping binding sites on the ribosome . Addition of release factor RF3 to the translation system abolishes the inhibitory effect of high RF1 concentration and increases the overall rate of ribosome recycling . These data can be explained by a three-step model for termination where the first step is RF1-promoted hydrolysis of peptidyl-tRNA . The second step is an intrinsically slow dissociation of RF1 which is accelerated by RF3 . The third step, catalysed by RRF and elongation factor G, leads to mobility of the ribosome on mRNA allowing it to enter a further round of translation . In the absence of RF3, RF1 can re-associate rapidly with the ribosome after peptidyl-tRNA hydrolysis, preventing RRF from entering the ribosomal A-site and thereby inhibiting ribosomal recycling . The overproduction of RF1 in cells deficient in RRF or lacking RF3 has effects on growth rate predicted by the in vitro experiments .

Arch Biochem Biophys, 1997 Nov 1, 347(1), 148 - 54
Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain; Kawashima H et al.; We previously reported the cDNA cloning of three new forms of P450, CYP4F4, CYP4F5, and CYP4F6, from a rat brain cDNA library . In the present study, we expressed CYP4F4 and CYP4F5 in Escherichia coli using the pCWOri expression vector with a modification of their N-terminal amino acid sequences and the incorporation of a C-terminal {His}4 tag to aid in purification . CYP4F5 recombinant protein was purified to a specific content of 7.7 nmol/mg protein from the membrane fraction of E . coli and showed omega-hydroxylation activity toward leukotriene B4 (LTB4), a chemical mediator of inflammation . On the other hand, the solubilized membrane fraction of CYP4F4-expressed recombinant protein catalyzed the omega-hydroxylation of prostaglandin A1, prostaglandin E1, and 6-trans-LTB4 as well as LTB4 . The effects of the peroxisome proliferator, clofibrate, on mRNA expression of CYP4F4, 4F5, and 4F6 were studied by Northern blot analysis . The expression levels of the mRNA of these CYP4Fs were shown to be reduced by clofibrate in liver .

Arch Biochem Biophys, 1997 Nov 1, 347(1), 103 - 12
Multiple levels of regulation of Escherichia coli succinyl-CoA synthetase; Birney M et al.; Concentrations of GDP, which are expected to bind to the catalytic site and inhibit the autophosphorylation of succinyl-CoA synthetase (SCS) when NTP is used as a substrate, were found to increase the level of phosphoenzyme formed . The ability of GDP to do so is dependent upon the presence of a protein distinct from SCS . The effector protein could be separated from SCS by ammonium sulfate fractionation . Reconstitution experiments show that the protein inhibits SCS, that the inhibition is relieved by GDP, and that the inhibitor recognizes both Escherichia coli and eukaryotic forms of SCS . The inhibitor is itself regulated by the conditions used to grow the bacteria and in a manner that appears distinct from that of SCS .

Arch Biochem Biophys, 1997 Nov 1, 347(1), 93 - 102
Escherichia coli flavodoxin sepharose as an affinity resin for cytochromes P450 and use to identify a putative cytochrome P450c17/3beta-hydroxysteroid dehydrogenase interaction; Jenkins CM et al.; Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E . coli culture) recombinant E . coli Fld, for use as an affinity resin for microsomal cytochromes P450 . As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins . Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation . Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis . Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose . Purified bovine 3beta-HSD covalently linked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen . This interaction, however, does not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro . From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum .

Arch Biochem Biophys, 1997 Nov 1, 347(1), 9 - 18
Molecular cloning, sequencing, and expression in Escherichia coli of mouse flavin-containing monooxygenase 3 (FMO3): comparison with the human isoform; Falls JG et al.; The sequence of mouse flavin-containing monooxygenase 3 (FMO3) was obtained from several clones isolated from a mouse liver cDNA library . The nucleotide sequence of mouse FMO3 was 2020 bases in length containing 37 bases in the 5' flanking region, 1602 in the coding region, and 381 in the 3' flanking region . The derived protein sequence consisted of 534 amino acids including the putative flavin adenine dinucleotide and NADP+ pyrophosphate binding sites (characteristic of mammalian FMOs) starting at positions 9 and 191, respectively . The mouse FMO3 protein sequence was 79 and 82% identical to the human and rabbit FMO3 sequences, respectively . Mouse FMO3 was expressed in Escherichia coli and compared to E . coli expressed human FMO3 . The FMO3 proteins migrated with the same mobility ( approximately 58 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting . The expressed FMO3 enzymes (mouse and human forms) were sensitive to heat and reacted in a similar manner toward metal ions and detergent . Catalytic activities of mouse and human FMO3 were high toward the substrate methimazole; however, in the presence of trimethylamine and thioacetamide, FMO-dependent methimazole oxidation by both enzymes was reduced by greater than 85% . Other substrates which inhibited methimazole oxidation were thiourea and thiobenzamide and to a lesser degree N,N-dimethylaniline . When probed with mouse FMO3 cDNA, FMO3 transcripts were detected in hepatic mRNA samples from female mice, but not in samples from males . FMO3 was detected in mRNA samples from male and female mouse lung, but FMO3 message was not detected in mouse kidney sample from either gender . Results of immunoblotting confirmed the tissue- and gender-dependent expression of mouse FMO3 .

Anal Biochem, 1997 Oct 15, 252(2), 299 - 307
A protease-free assay for peptidyl prolyl cis/trans isomerases using standard peptide substrates; Janowski B et al.; Peptidyl prolyl cis/trans isomerases (PPIases) are ubiquitous and abundant enzymes catalyzing peptide bond cis/trans isomerization adjacent to proline in peptides and proteins . An uncoupled protease-free assay of PPIase activity has been developed using the standard tetrapeptide substrates of the proteolytically coupled test system . Differences in the UV/vis absorption spectra of cis and trans conformations of Suc-Ala-Xaa-Pro-Phe-(Y-) anilide (Xaa = Ala, Leu, Phe; Y = 4-nitro, 2,4-difluoro) were exploited to monitor the time course of the cis/trans isomerization subsequent to a solvent jump from 0.47 M LiCl/trifluoroethanol into aqueous solution . The utility of the assay has been demonstrated by the determination of the Michaelis-Menten constants of cytosolic cyclophilin (Cyp18) and of the proteolytically sensitive FK506-binding protein-like PPIase SlyD from Escherichia coli . Furthermore, similar inhibition constants were estimated for the reversible inhibition of human Cyp18 by cyclosporin A (CsA) with both the proteolytically coupled and the novel uncoupled PPIase assay .

Anal Biochem, 1997 Oct 15, 252(2), 217 - 28
BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip; Nieba L et al.; While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system . We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag . This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here . The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal . Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface . Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH . In contrast, two His tags are usually found to be sufficient for stable binding . The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands . The sensor chip can be reused many times, because of the powerful regeneration methods . The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error .

Mol Endocrinol, 1997 Oct, 11(11), 1728 - 36
Purification, molecular cloning, and functional expression of the human nicodinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein; Vie MP et al.; The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding . THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides . Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin . This protein is a major structural kangaroo lens protein with no known function in other species . A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA . The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase . The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein . Purified GST fusion protein, but not GST, bound T3 specifically with high affinity {dissociation constant (Kd) = 0.5 nM} in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized {(125)I}T3 . T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH {activation constant (K{act}) = 10(-8) M}, but not by NADH . The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.

Arch Biochem Biophys, 1997 Oct 1, 346(1), 125 - 30
Expression and purification of recombinant rhinovirus 14 3CD proteinase and its comparison to the 3C proteinase; Davis GJ et al.; Human rhinovirus (HRV) is a positive-stranded RNA virus with an open reading frame that encodes for a single polyprotein of about 3000 amino acids . The HRV polyprotein is proteolytically processed; eight of nine cleavages are catalyzed by the 3C and/or the 3CD proteinases . We have expressed and purified recombinant HRV14 3C and 3CD proteinases and investigated their substrate selectivity and inhibitor sensitivity . Expressed 3CD proteinase had the P1/P1' residues of the 3C/3D cleavage site mutated from Gln/Gly to Ala/Ala in order to prevent autocleavage . The 3CD proteinase activities were measured by utilization of native, chromogenic, and fluorogenic peptide substrates . The 3CD proteinase exhibited < or =15% activity, compared to 3C, toward peptidyl p-nitroanilide substrates which contain only the p-nitroaniline moiety in the prime side . The 3C and 3CD proteinases exhibited similar activities for both internally quenched fluorogenic and native peptides . These results suggest that the two enzymes have similar but slightly different substrate specificity, especially on their preference for prime side residues . Inhibitor sensitivities toward classical proteinase inhibitors were generally similar for both enzymes . Small peptidyl inhibitors, specifically designed and synthesized for HRV14 3C, also inhibited the 3CD proteinase . Taken together, our data indicate that the 3D domain of 3CD proteinase had some influence on substrate recognition, but did not have dramatic impact on its interaction with inhibitors.

Arch Biochem Biophys, 1997 Oct 1, 346(1), 113 - 24
Expression cloning of a novel farnesylated protein, RDJ2, encoding a DnaJ protein homologue; Andres DA et al.; The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in cellular proteins which terminate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is most often methionine or serine . Substrates include the p21ras proteins, nuclear lamins, and a series of retinal proteins . To date, a limited number of substrates for the farnesyltransferase have been identified, predominantly by demonstration of the attachment of a farnesyl group to previously identified cDNA clones which encode proteins containing an appropriate carboxyl-terminal tetrapeptide . We describe here the use of a cDNA fusion protein expression library, together with enzymatic in vitro {3H}farnesyl radiolabeling, as a means of identifying novel farnesylated proteins . One candidate cDNA was fully cloned and found to be a homologue of the Escherichia coli heat shock gene dnaJ . The predicted amino acid sequence of this protein was found to terminate with the tetrapeptide Cys-Ala-His-Gln, which conforms to the consensus sequence for recognition by farnesyltransferase, and was shown to undergo in vivo farnesylation . This farnesylated protein, designated RDJ2 (rat DnaJ homologue 2), is a novel and ubiquitously expressed DnaJ homologue and is the newest member of the subfamily of DnaJ-related proteins which are posttranslationally modified by protein farnesylation.

Appl Environ Microbiol, 1997 Oct, 63(10), 4083 - 6
Survival of Escherichia coli cells exposed to iodoacetate and chlorodinitrobenzene is independent of the glutathione-gated K+ efflux systems KefB and KefC; Ness LS et al.; The KefB and KefC systems of Escherichia coli cells are activated by iodoacetate (IOA) and chlorodinitrobenzene (CDNB), leading to a rapid drop in the intracellular pH . However, survival of exposure to IOA or CDNB was found to be essentially independent of KefB and KefC activation . No correlation was found between the toxicity of the compound and its ability to elicit protective acidification via activation of KefB and KefC.

Mol Biotechnol, 1997 Aug, 8(1), 53 - 60
Molecular cloning of PCR fragments with cohesive ends; Delidow BC; Use of the polymerase chain reaction (PCR) provides a convenient means of generating DNA fragments for insertion into plasmids . Large quantities of the desired insert, bounded by convenient restriction sites, may be synthesized . The primers are chosen to span a known region of interest, and extended at their 5'-ends to include the desired restriction sites . Amplification of the target sequence is followed by precipitation of the product with ammonium acetate and ethanol to remove the primers . A small amount of product is analyzed by gel electrophoresis to ensure correct amplification, the remainder is digested with the appropriate restriction enzyme(s) . Restricted insert DNA is added to similarly restricted plasmid DNA in several ratios and incubated with DNA ligase to recircularize . Ligation products are used to transform competent bacteria . Clones containing inserts are identified by restriction digestion of plasmid minipreps from bacterial colonies.

Mol Biotechnol, 1997 Aug, 8(1), 1 - 6
A novel method for the purification of recombinant subunit I of the Dolichos biflorus seed lectin; Deb S et al.; Lectins are carbohydrate-binding proteins that are ubiquitous in nature . Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques . Plant lectins are one of the most thoroughly studied class of lectins, however, details of their in situ function remains elusive . Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous overexpression . The larger subunit of the Dolichos biflorus seed lectin was described by Chao et al . in 1994 and purification through affinity chromatography techniques was described . Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars . This method may have uses in the purification of mutant proteins that may not bind carbohydrates . Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090 +/- 16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.

J Pediatr Gastroenterol Nutr, 1997 Oct, 25(4), 394 - 9
Colonic structural and ion transport abnormalities in suckling rabbits infected with Escherichia coli K12; O'Loughlin EV et al.; BACKGROUND: Escherichia coli K12 is a laboratory strain considered nonpathogenic . The purpose of this study was to examine the effect of E . coli K12 infection on colonic structure and function . METHODS: Suckling rabbits were infected at 10 days of age with 6 x 10(9) CFU E . coli by intragastric inoculation and were examined 4 to 5 days later . Segments of ileum and proximal and distal colon were removed for light and electron microscopy, and NaCl transport was examined in vitro under short-circuited conditions in Ussing chambers . RESULTS: Infection did not cause weight loss or diarrhea . Colonic mucosa was inflamed with infiltration by polymorphonuclear neutrophils mainly in the lamina propria . The proximal and distal colon exhibited reduced Na+ absorption . The proximal colon also showed increased Cl- secretion; the ileum was unaffected . CONCLUSIONS: Infection with E . coli K12 disrupts the epithelium and alters ion transport in the colon, probably as a result of mucosal inflammation . The changes indicate that nonpathogenic E . coli have the potential to cause intestinal disease.

Bioconjug Chem, 1997 Sep-Oct, 8(5), 743 - 50
Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking; Tagge E et al.; We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC . The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence . Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 degrees C and, on incubation at 37 degrees C, distributed initially to endosomes and then to Golgi vesicles . Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing . Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured . Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells . Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells . These data support a model requiring a Golgi-dependent step in cell intoxication by ricin . The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.

Bioconjug Chem, 1997 Sep-Oct, 8(5), 702 - 7
Water-soluble polyion complex associates of DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer; Katayose S et al.; Complex formation of poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) AB type block copolymer with salmon testes DNA or Col E1 plasmid DNA in aqueous milieu was studied . The PLL segment of PEG-PLL interacts with nucleic acid through an electrostatic force to form a water-soluble complex associate with a diameter of ca . 50 nm . PEG segments surrounding the core of the polyion complex prevented the complex from precipitation even under stoichiometric conditions, at which the unit ratio of L-lysine in PEG-PLL and phosphate in the DNA is equal . The profile of the thermal melting curve revealed a higher stabilization of DNA structure in PEG-PLL/DNA complexes compared to that in the complex made from DNA and PLL homopolymer with the same molecular weight as the PLL segment in PEG-PLL . This stabilizing effect on the DNA structure may be due to the compartmentalization of DNA into the microenvironment of PEG with low permittivity . The reversible nature of the PEG-PLL/DNA complex was further verified through the addition of polyanion {poly-(L-aspartic acid)}: Poly(L-aspartic acid) replaced DNA in the complex with PEG-PLL, resulting in the release of free DNA in the medium . Furthermore, the PEG-PLL/DNA complex showed high resistance against DNase I attack, suggesting DNA protection through the segregation into the core of the associate having PEG palisade.

RNA, 1997 Oct, 3(10), 1173 - 81
Mammalian prothymosin alpha links to tRNA in Escherichia coli cells; Vartapetian A et al.; Prothymosin alpha, a small and highly acidic nuclear protein related to cell proliferation, is known to be covalently attached to a small unidentified cytoplasmic RNA in mammalian cells . Here we demonstrate that recombinant rat prothymosin a links covalently to an RNA when overproduced in Escherichia coli cells . The RNA species of this complex is represented by a wide range of bacterial tRNAs . tRNA(Lys), tRNA(3Ser), tRNA(2Ile), and tRNA(mMet) were identified by sequencing . Prothymosin alpha appears to be linked to the 5' terminus of tRNA . tRNA attachment site lies close to the carboxy-terminus of prothymosin alpha . Furthermore, the carboxy-terminal peptide of prothymosin alpha is also competent for tRNA binding . The site of tRNA attachment coincides with the nuclear localization signal of prothymosin alpha, and tRNA binding might be expected to affect subcellular localization of this protein.

Arch Microbiol, 1997 Nov, 168(5), 421 - 7
The path of unspecific incorporation of selenium in Escherichia coli; Muller S et al.; The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation . Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically . Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis . On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels . These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E . coli occurs mainly as selenocysteine . These findings also provide information on the labeling conditions for incorporating 75Se only and specifically into selenoproteins.

Arch Microbiol, 1997 Nov, 168(5), 412 - 20
Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus; Steen IH et al.; A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized . The mol . mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE . Following separation by SDS-PAGE, A . fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining . The enzyme showed dual coenzyme specificity with a high preference for NADP+ . Optimal temperature for activity was 90 degrees C or above, and a half-life of 22 min was found for the enzyme when incubated at 90 degrees C in a 50 mM Tricine-KOH buffer (pH 8.0) . Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned . DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme . The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli . All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E . coli isocitrate dehydrogenase are conserved in the enzyme from A . fulgidus . The primary structure of A . fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea . Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.

Arch Microbiol, 1997 Nov, 168(5), 403 - 11
Characterisation of Escherichia coli K-12 mutants defective in formate-dependent nitrite reduction: essential roles for hemN and the menFDBCE operon; Tyson K et al.; Three Escherichia coli mutants defective in formate-dependent nitrite reduction (Nrf activity) were characterised . Two of the mutants, JCB354 and JCB356, synthesized all five c-type cytochromes previously characterised in anaerobic cultures of E . coli . The third mutant, JCB355, was defective for both cytochrome b and cytochrome c synthesis, but only during anaerobic growth . The insertion sites of the transposon in strains JCB354 and JCB356 mapped to the menFDBCE operon; the hemN gene was disrupted in strain JCB355 . The mutation in strain JCB354 was complemented by a plasmid encoding only menD; strain JCB356 was complemented by a plasmid encoding only menBCE . A mutant defective in the methyltransferase activity involved in both ubiquinone synthesis and conversion of demethylmenaquinone to menaquinone expressed the same Nrf activity as the parental strain . The effects of men, ubiA and ubiE mutations on other cytochrome-c-dependent electron transfer pathways were also determined . The combined data establish that menaquinones are essential for cytochrome-c-dependent trimethylamine-N-oxide reductase (Tor) and Nrf activity, but that either menaquinone or ubiquinone, but not demethylmenaquinone, can transfer electrons to a third cytochrome-c-dependent electron transfer chain, the periplasmic nitrate reductase.

J Biol Chem, 1997 Oct 10, 272(41), 25935 - 40
Thioltransferase (glutaredoxin) is detected within HIV-1 and can regulate the activity of glutathionylated HIV-1 protease in vitro; Davis DA et al.; Previous studies have suggested that the two conserved cysteines of the HIV-1 protease may be involved in regulating protease activity . Here, we examined diglutathionylated wild type protease (Cys-67-SSG, Cys-95-SSG) and the monoglutathionylated protease mutants (C67A, Cys-95-SSG and C95A, Cys-67-SSG) as potential substrates for thioltransferase (glutaredoxin) . Time-dependent changes in the extent of deglutathionylation of each protein were assayed by reverse phase-high performance liquid chromatography . Glutathione alone was not an effective reductant, whereas thioltransferase displayed differential catalysis toward the Cys-95-SSG and Cys-67-SSG sites . At low thioltransferase concentrations (5 nM), deglutathionylation occurred almost exclusively at Cys-95-SSG . With substantially more thioltransferase (100 nM) Cys-67-SSG was partially deglutathionylated but only at 20% of the rate of Cys-95-SSG reduction . Treatment of the diglutathionylated protease with thioltransferase not only restored protease activity but generated an enzyme preparation that had a 3- to 5-fold greater specific activity relative to the fully reduced form . Immunoblot analysis of HIV-1MN virus with an antibody to thioltransferase detected a band co-migrating with recombinant thioltransferase that persisted following subtilisin treatment, indicating the presence of thioltransferase within HIV-1 . Our results implicate thioltransferase in the regulation and/or maintenance of protease activity in HIV-1 infected cells.

J Biol Chem, 1997 Oct 10, 272(41), 25907 - 12
Caveolin versus calmodulin . Counterbalancing allosteric modulators of endothelial nitric oxide synthase; Michel JB et al.; Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases . The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae . Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J . B., Feron, O., Sacks, D., and Michel, T . (1997) J . Biol . Chem . 272, 15583-15586) . Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain . Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli . The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin . These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

J Biol Chem, 1997 Oct 10, 272(41), 25802 - 8
Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit; Vilei EM et al.; The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system . The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively . The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol . In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it . Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system . While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively . Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis . A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells . This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested . Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations . All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins . Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.

J Biol Chem, 1997 Oct 10, 272(41), 25761 - 7
Catalytic and DNA binding properties of PvuII restriction endonuclease mutants; Nastri HG et al.; The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches . While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results . Aspartate 34, which contacts the central base pair of the PvuII site (5'-CAGCTG-3') through the minor groove, plays a critical role in binding specificity . A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site . In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection . Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA- or subunit-subunit contacts rather than in the catalytic center . This provides further evidence for a strong linkage between specific binding and catalysis.

J Biol Chem, 1997 Oct 10, 272(41), 25719 - 23
Biochemical characteristics of caspases-3, -6, -7, and -8; Stennicke HR et al.; The observation that the nematode cell death effector gene product Ced-3 is homologous to human interleukin-1beta-converting enzyme (caspase-1) has led to the discovery of at least nine other human caspases, many of which are implicated as mediators of apoptosis . Significant interest has been given to aspects of the cell biology and substrate specificity of this family of proteases; however, quantitative descriptions of their biochemical characteristics have lagged behind . We describe the influence of a number of environmental parameters, including pH, ionic strength, detergent, and specific ion concentrations, on the activity and stability of four caspases involved in death receptor-mediated apoptosis . Based on these observations, we recommend the following buffer as optimal for investigation of their characteristics in vitro: 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% 3-{(3-cholamidopropyl)dimethylammonio}-2-hydroxy-1-propanesulfonic acid (CHAPS), 10% sucrose, pH 7.2 . Caspase activity is not affected by concentrations of Ca2+ below 100 mM, but is abolished by Zn2+ in the submicromolar range, a common characteristic of cysteine proteases . Optimal pH values vary from 6.8 for caspase-8 to 7.4 for caspase-3, and activity of all is relatively stable between 0 and 150 mM NaCl . Consequently, changes in the physiologic pH and ionic strength would not significantly alter the activity of the enzymes, inasmuch as all four caspases are optimally active within the range of these parameters found in the cytosol of living and dying human cells.

J Biol Chem, 1997 Oct 10, 272(41), 25678 - 84
Isolation from phage display libraries of single chain variable fragment antibodies that recognize conformational epitopes in the malaria vaccine candidate, apical membrane antigen-1; Fu Y et al.; Phage display of single chain variable fragment (scFv) antibodies is a powerful tool for the selection of important and useful antibody specificities . We have constructed such a library from mice protected from malaria challenge by immunization with recombinant Plasmodium chabaudi DS apical membrane antigen (AMA-1) . Panning on refolded AMA-1 enriched a population of scFvs which specifically bound the antigen . The single chain antibodies recognize conformational epitopes on AMA-1 from the P . chabaudi DS strain but not on AMA-1 of the 556KA strain of P . chabaudi . A subset of the antibody fragments recognized AMA-1 from the human malaria parasite Plasmodium falciparum . Nucleotide sequencing revealed that at least four unique scFv genes were selected by the panning procedure . These scFv antibodies are valuable reagents for probing the structure and function of AMA-1 and will be used to test the feasibility of using recombinant antibodies in a passive immunization therapy against malaria.

J Biol Chem, 1997 Oct 10, 272(41), 25573 - 5
A mechanism for complementation of the sodA sodB defect in Escherichia coli by overproduction of the rbo gene product (desulfoferrodoxin) from Desulfoarculus baarsii; Liochev SI et al.; Overexpression of rbo in Escherichia coli prevents the inactivation of the {4Fe-4S}-containing fumarases that otherwise occurs in the sodA sodB strain . It similarly protects against the increased sensitivity toward H2O2, which is imposed by the lack of SOD A and SOD B . These results would be explained on the basis of scavenging of O-2 within the cells by RBO . This interpretation was supported by measurements of intracellular scavenging of O-2 by the lucigenin luminescence method . Since SOD activity could not be detected in dilute extracts, of the RBO-overexpressing sodA sodB strain, we propose that RBO catalyzes the reduction of O-2 at the expense of cellular reductants such as NAD(P)H . A similar mechanism may apply to other instances of complementation of SOD defects by non-SOD genes.

J Biol Chem, 1997 Oct 10, 272(41), 25560 - 5
Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle; Baek SH et al.; A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method . Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs revealed that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and is related to members of the UBP family containing highly conserved Cys and His domains . Chick UBP41 was expressed in E . coli and purified from the cells to apparent homogeneity, using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate . The purified enzyme behaved as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain . Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide . The UBP41 protease cleaved at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes; thus, it is active against ubiquitin-beta-galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids . UBP41 also released free ubiquitin from poly-His-tagged di-ubiquitin . Moreover, it converted poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules were not further degraded to free ubiquitin and lysozyme . These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.

J Biol Chem, 1997 Oct 10, 272(41), 25531 - 6
Human low density lipoprotein receptor fragment . Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli; Simmons T et al.; The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE . The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds . As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion . Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions . We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency . The active fragment was isolated and purified with an LDL affinity column . The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing . The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked . In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3) . Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors . We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.

J Biol Chem, 1997 Oct 10, 272(41), 25483 - 92
Characterization of recombinant REGalpha, REGbeta, and REGgamma proteasome activators; Realini C et al.; Full-length cDNAs for three human proteasome activator subunits, called REGalpha, REGbeta, and REGgamma, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized . Recombinant alpha or gamma subunits form heptameric species; recombinant beta subunits are found largely as monomers or small multimers . Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes . The pattern of activated peptide hydrolysis is virtually identical for REGalpha and REGbeta . These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides . Recombinant alpha and beta subunits bind each other with high affinity, and the REGalpha/beta heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-beta-nitroanilide (LLE-betaNA) more than REGalpha or REGbeta alone . Using filter binding and gel filtration assays, recombinant REGgamma subunits were shown to bind themselves but not alpha or beta subunits . REGgamma differs from REGalpha and REGbeta in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-betaNA by the proteasome . REGgamma binds the proteasome with higher affinity than REGalpha or REGbeta yet with lower affinity than complexes containing both REGalpha and REGbeta . In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.

J Biol Chem, 1997 Oct 10, 272(41), 25437 - 40
Dissecting the interaction between nitric oxide synthase (NOS) and caveolin . Functional significance of the nos caveolin binding domain in vivo; Garcia-Cardena G et al.; Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells . Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins . To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo . Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins . Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178) . Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities . These results suggest a common mechanism and site of inhibition . Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570 . Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments . Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS . This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells . Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Virology, 1997 Sep 29, 236(2), 364 - 73
Characterization of the reverse transcriptase of a human immunodeficiency virus type 1 group O isolate; Quinones-Mateu ME et al.; The catalytic properties and sensitivity to different inhibitors have been determined for the reverse transcriptase (RT) of group O human immunodeficiency virus type 1 (HIV-1) . The RT-coding region was cloned from a new HIV-1 group O isolate from Spain, expressed in Escherichia coli, and purified by affinity chromatography . This new RT showed 79% amino acid sequence identity with the corresponding enzyme of group M subtype B strain BH10 . The two enzymes showed very similar kinetics of RNA-dependent DNA polymerization using homopolymeric template-primers and RNase H specific activity . Inhibitor sensitivity to ddTTP and 3'-azido-2',3'-dideoxythymidine triphosphate (AZTTP) was also similar for both enzymes . However, the two enzymes differed dramatically in their sensitivity to several inhibitors . While the RT of the BH10 isolate was sensitive to nevirapine and loviride (IC50 ranged from 0.16 to 8.2 microM, depending on the substrates used), the enzyme of the Spanish HIV-1 group O isolate showed high-level resistance to those compounds (IC50 > 200 microM) . The amino acid sequence of the RT of group O HIV-1 contains three amino acids (Cys-181, Glu-179, and Gly-98), which are found in group M subtype B strains resistant to nonnucleoside RT inhibitors . The recombinant group O HIV-1 RT should be useful for studies aimed at discovering and designing drugs directed toward group O isolates of HIV-1.

Protein Expr Purif, 1997 Oct, 11(1), 125 - 34
High level expression and characterization of recombinant human hippocampus phenol sulfotransferase: a novel phenol-sulfating form of phenol sulfotransferase; Hwang SR et al.; Phenol sulfotransferases (PSTs) represent a family of sulfotransferase enzymes that modify the biologic activities and excretion of phenolic compounds and monoamines . A novel human hippocampal PST (H-PST) cDNA with homology to phenol (P) and monoamine (M) forms of PST was previously isolated from brain . To compare the biochemical properties of H-PST with that of phenol (P-PST) and monoamine (M-PST) sulfotransferases, high level expression of recombinant H-PST was achieved in this study with the pET3c vector in BL21(DE3) Escherichia coli cells . Expression was demonstrated by isopropyl beta-D-thiogalactopyranoside induction of 34-kDa H-PST that represented 5-10% of total E . coli proteins . Purification by ion-exchange chromatography on DEAE-Sepharose yielded more than 2 mg of H-PST . Characterization showed that H-PST exists as a homodimer of 60-65 kDa by gel filtration chromatography . H-PST prefers p-nitrophenol as substrate and does not sulfate dopamine or neuropeptide substrates . Kinetic studies showed that H-PST possessed K(m(app)) and Vmax(app) values of 3 microM p-nitrophenol and 160 nmol/min/mg, respectively . H-PST was sensitive to inhibition by DCNP (2,6-dichloro-4-nitrophenol) . H-PST is thermolabile since its activity was reduced upon preincubation at 37 degrees C . These results indicate that H-PST shows similarities and differences compared to P-PST and M-PST sulfotransferases . P-PST prefers p-nitrophenol as substrate, is sensitive to inhibition by DCNP, and is thermostable; in contrast, M-PST prefers monoamines as substrate, is not sensitive to DCNP, and is thermolabile . The distinct profile of biochemical properties of H-PST, and its primary sequence homology to P-PST and M-PST, suggests that H-PST represents a novel allelic variant of human phenol sulfotransferases . Importantly, this study demonstrates that high level expression of H-PST allows determination of distinguishing characteristics of variant forms of PSTs.

Protein Expr Purif, 1997 Oct, 11(1), 111 - 8
Affinity purification and elimination of methionine oxidation in recombinant human cystatin C; Berti PJ et al.; Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide {Met(O)} residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature . Oxidation occurred in the periplasmic space or intracellularly during aerobic expression . Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry . Oxidation of Met110 was not observed . Growth under anaerobic conditions and modified purification procedures prevented oxidation . Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-{N-{(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl}amino}-4-g uanidinobutane), with elution with sodium trichloroacetate . The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively . This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged . The NMR observation of small, localized conformational changes was consistent with this . (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.

Protein Expr Purif, 1997 Oct, 11(1), 79 - 85
Labeling of recombinant protein for NMR spectroscopy: global and specific labeling of the rat liver fructose 2,6-bisphosphatase domain; Okar DA et al.; Methods for the efficient use of the 13C-labeled nutrients, glucose and histidine, in the production of recombinant protein were developed to provide the large amount of sample required for NMR studies . The nutrient requirements were reduced by determining the minimum amount of these metabolites needed during both the growth and the induction phases of the BL21(DE3) and newly constructed BL21(DE3) histidine auxotrophic Escherichia coli cultures . These methods were developed using the separate bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase, which is expressed to high levels in the pET3a/BL21 (DE3) bacterial system . Use of the optimized expression methods reduced the requirements for the labeled nutrients, glucose and histidine, by 90 and 93.8%, respectively . The savings realized by use of the minimized media and modified induction protocols were obtained without significant reduction of the yield of purified protein . Comprehensive study of the bisphosphatase domain by NMR spectroscopy requires large amounts of protein because of its low solubility and the short lifetime (2-3 days) of the NMR samples . The significant reduction in the costs of labeled protein samples realized by the optimized expression methods can meet these sample requirements in a cost-effective way, and thereby, allow NMR studies of the bisphosphatase domain to proceed.

Protein Expr Purif, 1997 Oct, 11(1), 53 - 60
Quantitative evaluation of neurotensin receptor purification by immobilized metal affinity chromatography; Grisshammer R et al.; Immobilized metal affinity chromatography has recently been used for purification of histidine-tagged membrane proteins in the presence of detergents with varying success . Strong binding to the metal resin is essential for purification when expression levels are low . We have investigated the influence of tag length and type of detergent on the purification of a neurotensin receptor fusion protein expressed in Escherichia coli at a level of about 0.1% of membrane protein . Receptors with six C-terminal histidine residues did not bind to nickel resin in the presence of the anionic detergent sodium dodecyl sulfate . In contrast, partial purification assessed by densitometry of Coomassie-stained gels was achieved using the nonionic detergents dodecyl maltoside or Triton X-100 (53% pure), or a detergent mixture containing the zwitterionic detergent 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate (46% pure) . Linking a highly charged epitope tag to the histidine tail did not affect the nickel-binding properties of receptors . The level of purification was substantially improved (72% pure) by extending the histidine tail to 10 residues because this allowed stringent washes at high imidazole concentration to remove nonspecifically bound contaminants . This strategy not only resulted in efficient purification of receptors from crude membranes, but also worked particularly well for single-step purification from total cell lysates, resulting in 340-fold purification of functional neurotensin receptor.

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