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: express