J Chromatogr A, 2000 Dec 29, 904(2), 145 - 69 Comparative studies of recombinant human granulocyte-colony stimulating factor, its Ser-17 and (His)6-tagged forms interaction with metal ions by means of immobilized metal ion affinity partitioning . Effect of chelated nickel and mercuric ions on extraction and refolding of proteins from inclusion bodies; Zaveckas M et al.; The chelation capability of the reactive dye Light Resistant Yellow 2KT towards metal ions, particularly mercury(II) was evaluated in the pH range 5.0-7.0, and it was shown that the dye-Hg(II) complex has a free site for the interaction with human recombinant granulocyte-colony stimulating factor (rhG-CSF) from Escherichia coli . Affinity partitioning of three rhG-CSF forms--native, rhG-CSF{Cys17--->Ser17} and (His)6-rhG-CSF was studied in aqueous two-phase systems, which contained metal ions--Cu(II), Ni(II) and Hg(II)--chelated by dye-poly(ethylene glycol) at pH 5.0 and 7.0, in the presence or absence of many selected agents . It was determined, that chelated Ni(II) ions exhibited stronger interaction with the hexahistidine-tagged protein form, while the extraction power of Cu(II) ions was found to be of comparable order of magnitude for all three protein forms at pH 7.0 . A comparative study of rhG-CSF and both its forms partitioning in the presence of chelated Hg(II) ions at pH 7.0 and 5.0 revealed possible direct interaction between Hg(II) ions and unpaired Cys-17 of rhG-CSF . The partitioning of three rhG-CSF forms inclusion body extract was studied in the presence of chelated Ni(II) and Hg(II) ions thus explaining the efficiency of targeted proteins renaturation gained upon their inclusion body forms interactions with chelated metal ions. J Chromatogr A, 2000 Dec 29, 904(2), 131 - 43 Chelated mercury as a ligand in immobilized metal ion affinity chromatography of proteins; Gelunaite L et al.; Chelation of mercuric ions by an iminodiacetate-Sepharose gel was evaluated . The retentive properties of iminodiacetate-Sepharose gel column was studied towards proteins varying the composition of eluting systems from 2-mercaptoethanol to NaCl and imidazole, determining also the extent of mercury leaching . It was demonstrated that chelated mercury contained free sites for interaction with proteins such as bromelain and recombinant human granulocyte colony stimulating factor from E . coli . The extraction of the latter by chromatography of its inclusion bodies solution on Hg(II)-loaded Sepharose-iminodiacetate gel was also evaluated. Cell Transplant, 2000 Nov-Dec, 9(6), 773 - 83 Alginate-encapsulated producer cells: a potential new approach for the treatment of malignant brain tumors; Thorsen F et al.; In recent years gene therapy has evolved as a new treatment for brain tumors, where genetically engineered cells can be used to deliver specific substances to target cells . However, clinical success has been limited due to insufficient gene transfer, lack of prolonged gene expression, and immunorejection of producer cells . These obstacles may be overcome by encapsulating producer cells into immunoisolating substances such as alginate . This may provide a stable in situ delivery system of specific proteins, which can interfere with tumor growth and differentiation . This article represents a fundamental study describing the in vitro and the in vivo behavior of alginate-encapsulated producer cells . The viability and cell cycle distribution of encapsulated NIH 3T3 cells was studied by confocal laser scanning microscopy (CLSM) and by flow cytometry . The CLSM study showed a high viability of the encapsulated NIH 3T3 cells during 9 weeks in culture . The flow cytometric analysis revealed a change in cellular ploidy after 1 week in culture, with normalization in ploidy after 3 and 9 weeks . The production of the bacterial E . coli beta-galactosidase in alginate-encapsulated BT4CnVlacZ cells was studied by x-gal staining, and the cells expressed prolonged beta-galactosidase activity . H528 hybridoma cells producing monoclonal antibodies (mAbs) against the human epidermal growth factor receptor (EGFR) were encapsulated in alginate, and the mAb release was determined . The release of mAbs stabilized around 400 ng/ml/h after 12 days in vitro . To actually demonstrate that alginate-encapsulated H528 cells potentially inhibit a heterogeneous glioma cell population, cell migration from human GaMg glioma spheroids was studied during stimulation with EGF in the presence of encapsulated H528 cells . The migration in vitro was totally inhibited in the presence of H528 encapsulated cells . Alginate beads with H528 cells were also implanted into rat brains, and after 9 weeks the distribution of mAbs within the brain was studied by immunohistochemistry . It is shown that the alginate entrapped H528 cells produce mAbs inside the brain for prolonged periods and that the mAbs are distributed within all CSF compartments . Encapsulated producer cells represent a potential delivery system for specific proteins to brain tumors . Different producer cells may be encapsulated in alginate to target phenotypic features and microenvironmental factors, which may influence the progressive growth of brain tumors. Clin Experiment Ophthalmol, 2000 Dec, 28(6), 434 - 6 Coexistent adenoviral keratoconjunctivitis and Acanthamoeba keratitis; Gajdatsy AD et al.; A 17-year-old youth presented with bilateral follicular conjunctivitis and nummular subepithelial corneal infiltrates . Failure of this to settle in an outpatient setting led to corneal scraping with microscopy and culturing for bacteria, fungi, Herpes simplex, adenovirus and Acanthamoeba as an inpatient . Polymerase chain reaction analysis of corneal cells was positive for adenovirus, and culture on live Escherichia coli-coated agar plates was positive for Acanthamoeba by phase contrast microscopy on day two . We conclude that Acanthomoeba infection can complicate adenoviral keratoconjunctivitis . This observation is in keeping with previously reported modes of infection by Acanthamoeba, whereby any epithelial breach seems to allow inoculation of the eye by this opportunistic organism. Plant Mol Biol, 2000 Dec, 44(6), 711 - 21 Single amino acid substitutions at the acyl-CoA-binding domain interrupt 14{C}palmitoyl-CoA binding of ACBP2, an Arabidopsis acyl-CoA-binding protein with ankyrin repeats; Chye ML et al.; Cytosolic acyl-CoA-binding proteins (ACBPs) are small proteins (ca . 10 kDa) that bind long-chain acyl-CoAs and are involved in the storage and intracellular transport of acyl-CoAs . Previously, we have characterized an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP, designated ACBP1, demonstrating the existence of a new form of ACBP in plants (M.-L . Chye, Plant Mol . Biol . 38 (1998) 827-838) . ACBP1 likely participates in intermembrane lipid transport from the ER to the plasma membrane, where it could maintain a membrane-associated acyl pool (Chye et al., Plant J . 18 (1999) 205-214) . Here we report the isolation of cDNAs encoding ACBP2 (Mr 38,479) that shows conservation in the acyl-CoA-binding domain to previously reported ACBPs, and contains ankyrin repeats at its carboxy terminus . These repeats, which likely mediate protein-protein interactions, could constitute a potential docking site in ACBP2 for an enzyme that uses acyl-CoAs as substrate, in vitro binding assays on recombinant (His)6-ACBP2 expressed in Escherichia coli show that it binds 14{C}palmitoyl-CoA preferentially to 14{C}oleoyl-CoA . Analysis of the acyl-CoA-binding domain in ACBP2 was carried out by in vitro mutagenesis . Mutant forms of recombinant (His)6-ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA-binding domain were less effective in binding 14{C}palmitoyl-CoA . Northern blot analysis showed that the 1.6 kb ACBP2 mRNA, like that of ACBP1, is expressed in all plant organs . Analysis of the ACBP2 promoter revealed that, like the ACBP1 promoter, it lacks a TATA box suggesting the possibility of a housekeeping function for ACBP2 in plant lipid metabolism. Equine Vet J Suppl, 2000 Jun, (32), 26 - 31 Effect of eltenac in horses with induced endotoxaemia; MacKay RJ et al.; Ten horses were used in a crossover study to evaluate the effectiveness of eltenac against endotoxaemia . Eltenac (0.5 mg/kg bwt) or saline control was given i.v . then 15 min later, intravenous infusion of endotoxin was begun and continued for 120 min (total dose 100 ng/kg bwt) . Horses were monitored for heart and respiratory rates, pulmonary and carotid arterial pressure and core body temperature . Blood was sampled at intervals for measurement of haematological variables and plasma concentrations of lactate, prostanoid metabolites, tumour necrosis factor (TNF) and stress hormones . In comparison with saline-treatment, use of eltenac significantly protected against endotoxin-induced changes in respiratory rate, core temperature, systemic arterial blood pressure (SAP), pulmonary arterial pressure, PCV, and plasma protein, 6-keto prostaglandin F1 alpha, thromboxane B2, epinephrine, and cortisol concentrations . Despite statistical effect of eltenac on SAP, values in both treatment groups remained well above baseline throughout the evaluation period . Significant protective effect of eltenac was not found for heart rate, white blood cell count, plasma lactate concentration or TNF activity . On the basis of these results, it is expected that use of eltenac will provide clinical benefit in horses with naturally occurring endotoxaemia. J Clin Immunol, 2000 Nov, 20(6), 408 - 15 Complexes of serum amyloid P component and DNA in serum from healthy individuals and systemic lupus erythematosus patients; Sorensen IJ et al.; Serum amyloid P component (SAP) binds in vitro to DNA; based on findings in SAP-deficient mice it was proposed that SAP's role is to handle chromatin and DNA, thereby preventing formation of anti-DNA antibodies . For the first time we have shown the presence of Ca2+-dependent SAP-DNA complexes, measured by ELISA, in sera from both healthy volunteers and systemic lupus erythematosus patients (SLE) . The concentration of SAP-DNA complexes in SLE sera was significantly lower than in normal sera and particularly low in sera from patients with anti-DNA titers exceeding 50 . The complexes were dissociated by the SAP ligand heparin and were not demonstrable in EDTA plasma . Normal sera showed similar capacity to form SAP-DNA complexes with both thymus and Escherichia coli DNA, whereas significantly lower amounts of complexes, in particular with E . coli DNA, were formed in SLE sera . SLE patients with moderate to high anti-DNA titers showed a significant negative correlation between serum SAP's binding of E . coli DNA and the anti-DNA titer. Nature, 2001 Jan 18, 409(6818), 370 - 4 Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules; Dohoney KM et al.; Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme . RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein) . Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo . Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity . Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate . These observations place strong constraints on possible movement mechanisms . Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism. Nature, 2001 Jan 18, 409(6818), 366 - 70 A model for SOS-lesion-targeted mutations in Escherichia coli; Pham P et al.; The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E . coli pol III (ref . 4) . Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis . (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis . Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand . The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites. Biol Pharm Bull, 2001 Jan, 24(1), 14 - 8 Effects of substitution of conserved amino acid residues on the sugar-binding property of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans; Arata A et al.; The 32-kDa galectin (LEC-1) of the nematode Caenorhabditis elegans (C elegans) is composed of two tandemly repeated homologous sequences, each containing a carbohydrate-recognition domain (CRD) . Using the polymerase chain reaction (PCR) with LEC-1 cDNA as a template and "megaprimers", we performed site-directed mutagenesis to substitute conserved amino acid residues in these domains . The resultant mutated LEC-1s were produced in E . coli, and their binding abilities were estimated by affinity chromatography . When one of the conserved amino acid residues in the first lectin domain was substituted, the binding ability of the mutant protein to asialofetuin-agarose was reduced but still remained . The binding ability of such mutants was similar to that of the recombinant half molecule containing the second lectin domain (Ch) . However, when mutations were introduced into the second lectin domain, the binding ability of these mutant lectins to asialofetuin-agarose was significantly reduced just like the half recombinant molecule containing the first lectin domain (Nh) . The different effects of the substitution of amino acid residues on the two lectin domains suggest that the binding properties of the two sites are different and that LEC-1 acts as a "heterobifunctional crosslinker." Nucleosides Nucleotides Nucleic Acids, 2000 Oct-Dec, 19(10-12), 1885 - 909 Pyrophosphoryl derivatives of 1-(2-deoxy-3-O-phosphono-methyl-beta- and -alpha-D-erythro-pentofuranosyl)thymine: synthesis and substrate properties towards some DNA polymerases; Mikhailopulo IA et al.; The synthesis of 1-(2-deoxy-3-O-phosphonomethyl-beta-D-erythropentofuranosyl)thymine (17) and its alpha-anomer 18 is described . Attempts to prepare 1-{2-deoxy-3-O-(pyrophosphoryl)phosphonomethyl-beta-D-erythro-pentofuranosyl}thymine (19) by an activation of the respective phosphonate 17 with 1,1'-carbonyldiimidazole (Im2CO) resulted in the quantitative formation of the corresponding pyrophosphonate derivative 21 (Scheme 2) . Activation of inorganic pyrophosphate with Im2CO followed by the condensation with the phosphonates 17 and 18 afforded the desired analogues of nucleoside triphosphate 19 (35%) and its alpha-anomer 20 (27%) along with the respective pyrophosphonate derivatives 21 (37%) and 24 (38%) (Scheme 3) . It was found that compounds 19 and 20 display (i) no substrate properties toward calf thymus terminal deoxynucleotidyl transferase (TDT) and AMV reverse transcriptase, and (ii) moderate substrate activity with E . coli DNA polymerase I (Klenow fragment). J Mol Microbiol Biotechnol, 2001 Jan, 3(1), 135 - 42 Biosynthesis of K88 fimbriae in Escherichia coli: interaction of tip-subunit FaeC with the periplasmic chaperone FaeE and the outer membrane usher FaeD; Mol O et al.; K88 fimbriae are ordered polymeric protein structures at the surface of enterotoxigenic Escherichia coli cells . Their production and assembly requires a molecular chaperone located in the periplasm (FaeE) and a molecular usher located in the outer membrane (FaeD) . FaeC is the tip component of the K88 fimbriae . We studied the expression of the subcloned faeC gene, the subcellular localization of FaeC and its interaction with the chaperone and the outer membrane usher . In the absence of the chaperone or the usher, FaeC could not be detected in E . coli cells harbouring the faeC gene and its ribosome binding site under contol of the IPTG inducible lpp/lac promoter/operator . The expression of FaeC was detectable in the presence of chaperone FaeE, but a direct interaction between the chaperone and FaeC was not found . The expression of FaeC was also detectable in cells co-expressing the outer membrane usher FaeD . Overexpression of FaeC after changing the faeC ribosome binding site appeared to induce lethality . Expression of subcloned FaeC in the absence of FaeE or FaeD could be detected when faeC was cloned under the tight control of the ara promoter/operator and when lethality induction was avoided . The direct interaction of FaeC with outer membranes containing the usher FaeD was studied by cell fractionation, isopycnic sucrose density gradient centrifugation, SDS-PAGE and immunoblotting . FaeC was found to bind to outer membranes containing FaeD or a FaeD-PhoA hybrid construct containing 215 amino-terminal residues of FaeD . This binding was not observed when control outer membranes without FaeD were used . No other K88 specific proteins were required for this interaction . The direct interaction between FaeC and FaeD in the outer membranes was shown by affinity blotting experiments . FaeE was not required for this interaction . Together these data indicate that the minor fimbrial subunit FaeC, unlike FaeG, H and F, does not have a strong interaction with the chaperone FaeE in the E . coli periplasm, but directly binds to the outer membrane molecular usher FaeD. Parasitol Res, 2001 Jan, 87(1), 14 - 7 High prevalence of infection with Entamoeba dispar, but not E . histolytica, in captive macaques; Tachibana H et al.; A total of 268 nonhuman primates (20 species) kept in the Primate Research Institute, Kyoto University, Japan, were surveyed for intestinal amebas . Total positive rates as based on the presence of cysts in the stool following formalin-ether sedimentation were as follows: Entamoeba histolytica/E . dispar, 53%; E . coli, 34%; E . hartmanni, 34%; Iodamoeba buetschlii, 25%; Endolimax nana, 8%; and E . chattoni, 3% . Positive rates were higher in Old World monkeys and lower in New World monkeys . All the 141 E . histolytica/E . dispar-positive animals were Macaca monkeys . The E . histolytica/E . dispar-positive samples were analyzed by polymerase chain reaction (PCR) for identification of E . histolytica and E . dispar . E . dispar DNA was detected in 137 samples, whereas no E . histolytica DNA was seen . Zymodeme analysis and reactivity to monoclonal antibodies of cultured trophozoites also supported the presence of E . dispar and the absence of E . histolytica . When the sera of 93 macaques were examined by an indirect fluorescent antibody test, only 3 animals proved to be positive for E . histolytica, showing the lowest titer . These results demonstrate that infection with E . dispar, but not E . histolytica, is predominant in macaques. Antisense Nucleic Acid Drug Dev, 2000 Dec, 10(6), 463 - 8 A short phosphodiester window is sufficient to direct RNase H-dependent RNA cleavage by antisense peptide nucleic acid; Malchere C et al.; The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H . The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions . It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay. Plant Mol Biol, 2000 Nov, 44(5), 603 - 17 Molecular characterization of quinolinate phosphoribosyltransferase (QPRtase) in Nicotiana; Sinclair SJ et al.; Quinolate acid phosphoribosyltransferase (QPRTase), a key enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, also plays an important role in ensuring nicotinic acid is available for the synthesis of defensive pyridine alkaloids in Nicotiana species . In this study, cDNAs for QPRTase were characterized from N . rustica and N . tabacum . Deduced proteins from both cDNAs are almost identical and contain a 24 amino acid N-terminal extension, not reported in other QPRTases, that has characteristics of a mitochondrial targeting sequence . In N . tabacum and N . sylvestris, both of which contain nicotine as the major pyridine alkaloid, QPRTase transcript was detected in roots, the site of nicotine synthesis, but not in leaves . QPRTase transcript levels increased markedly in roots of both species 12-24 h after damage to aerial tissues, with a concomitant rise in transcript levels of putrescine N-methyltransferase (PMT), another key enzyme in nicotine biosynthesis . In N . glauca, however, in which anabasine represents the major pyridine alkaloid, QPRTase transcript was detected in both leaf and root tissues . Moreover, wound induction of QPRTase but not PMT was observed in leaf tissues, and not in roots, 12-24 h after wounding . Southern analysis of genomic DNA from the Nicotiana species noted above, and also several others from within the genus, suggested that QPRTase is encoded by a small gene family in all the species investigated. Plant Mol Biol, 2000 Nov, 44(5), 581 - 9 An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara; Newton SS et al.; Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes . The gene products participate either directly or indirectly towards increasing cold tolerance . Evidence of proteins having a direct effect on cold tolerance is emerging but limited . With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect . Purification of one such fraction resulted in isolation of a 25 kDa protein . N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins . When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at -8 degrees C . A cDNA library which was made in November from stems and leaves of S . dulcamara was successfully screened for the corresponding cDNA clone . The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues including a N-terminal signal sequence and a putative C-terminal propeptide . The mature protein, without the N-terminal signal sequence, was expressed in Escherichia coli . The partially purified protein in the supernatant fraction of the culture medium had cryoprotective activity. Shock, 2001 Jan, 15(1), 65 - 72 Differential roles of iNOS and nNOS at rostral ventrolateral medulla during experimental endotoxemia in the rat; Chan JY et al.; We investigated the differential contribution of inducible and neuronal nitric oxide synthase (iNOS and nNOS) at the rostral ventrolateral medulla (RVLM) to endotoxemia induced by E . coli lipopolysaccharide (LPS) . In Sprague-Dawley rats maintained under propofol anesthesia, i.v . administration of LPS (15, 30, or 45 mg/kg) induced a reduction (phase I), followed by an augmentation (phase II) and a secondary decrease (phase III) in the power density of the vasomotor components (0-0.8 Hz) in systemic arterial pressure (SAP) signals . LPS also induced an immediate hypotension, followed by a rebound increase and a secondary decrease in SAP . In addition, the level of iNOS mRNA exhibited a significant surge that began with phase I endotoxemia, reaching progressively its peak at phase III . Discernible down-regulation of nNOS mRNA was not detected until the last phase of endotoxemia . Pretreatment with microinjection of the selective iNOS inhibitor, aminoguanidine (250 pmol), into the bilateral RVLM significantly prolonged phases II and III endotoxemia, blunted the initial and secondary hypotension, and antagonized the upregulation of iNOS mRNA . Similar pretreatment with the selective nNOS inhibitor, 7-nitroindazole (1 pmol), on the other hand, discernibly shortened phase II and prolonged phase III endotoxemia, and induced progressive hypotension by antagonizing the rebound increase in SAP . We conclude that the relative prevalence of functional expression and molecular synthesis of iNOS over nNOS in the RVLM may be a crucial determinant for the reduction or loss in power density of the vasomotor components of SAP signals during experimental endotoxemia. J Nucl Med, 2001 Jan, 42(1), 117 - 23 Specific and rapid scintigraphic detection of infection with 99mTc-labeled interleukin-8; Rennen HJ et al.; Interleukin-8 (IL-8) is a chemotactic cytokine involved in activation and recruitment of neutrophils to areas of infection . In our previous studies in rabbits we tested 123I-labeled IL-8 for its potential to image infections and showed that IL-8 rapidly and efficiently accumulated in infectious foci . However, labeling of IL-8 with 123I is costly and laborious and the specific activity of the preparation was low . In this study IL-8 was labeled with 99mTc through the hydrazinonicotinamide (HYNIC) chelator . METHODS: The leukocyte receptor-binding capacity of the preparation was determined in vitro . Rabbits with Escherichia coli abscesses were injected intravenously with 7 MBq 99mTc-HYNIC-IL-8 . Biodistribution of the radiolabel was determined by gamma camera imaging and tissue counting at 8 h after injection . 99mTc-HYNIC-lysozyme was used as a size-matched control . RESULTS: The leukocyte receptor-binding capacity of the 99mTc-HYNIC-IL-8 preparation was preserved as determined in vitro, but labeling efficiency was modest with a specific activity of 3 MBq/microg . 99mTc-HYNIC-IL-8 accumulated rapidly in the abscess up to 0.33 +/- 0.06 percentage injected dose per gram (%ID/g) at 8 h after injection (vs . 0.025 +/- 0.003 %lD/g for 99mTc-HYNIC-lysozyme) . Total uptake in the abscess was 4.9 +/- 0.7 %ID (vs . 0.44 +/- 0.05 %ID for 99mTc-HYNIC-lysozyme) . Abscess-to-contralateral muscle ratios increased up to 127 +/- 23 (compared with 6.7 +/- 1.1 for 99mTc-HYNIC-lysozyme) and abscess-to-blood ratios increased to 11.9 +/- 2.2 (0.24 +/- 0.03 for 99mTc-HYNIC-lysozyme) . The radiolabel was excreted renally, with a retention in the kidneys of 28 %ID . Gamma camera imaging rapidly visualized the abscess from 1 h after injection onward, with abscess-to-background ratios improving with time up to 22 at 8 h after injection (vs . 2.7 for 99mTc-HYNIC-lysozyme), as determined by quantitative analysis of the images . Most important, only a transient (30 min) moderate drop of leukocyte counts and no leukocytosis were observed after injection of an imaging dose of 99mTc-HYNIC-IL-8 . CONCLUSION: IL-8 can be labeled with 99mTc using HYNIC as a chelator . By this method the leukocyte receptor-binding capacity is preserved . The preparation allows rapid visualization of infection in a rabbit model with high target-to-background ratios . The mild transient drop of leukocyte counts and the absence of leukocytosis suggest that 99mTc-HYNIC-IL-8 may be used as an imaging agent with only mild and transient side effects. Parasitology, 2001 Jan, 122 Pt 1, 1 - 13 Characterization of three genes encoding enzymes of the folate biosynthetic pathway in Plasmodium falciparum; Lee CS et al.; Although the folate metabolic pathway in malaria parasites is a major chemotherapeutic target, resistance to currently available antifolate drugs is an increasing problem . This pathway, however, includes a number of enzymes that, to date, have not been characterized despite their potential for clinical exploitation . As a step towards evaluation of additional targets in this pathway, we report the isolation and characterization of 3 new genes that encode homologues of GTP cyclohydrolase I (GTP-CH), dihydrofolate synthase/folylpolyglutamate synthase (DHFS/FPGS) and serine hydroxymethyltransferase (SHMT) . The genes encoding GTP-CH and SHMT are unambiguously assigned to chromosome 12, while that for DHFS/FPGS is tentatively assigned to chromosome 13 . All 3 genes are expressed in blood-stage parasites, yielding transcripts of which only ca 60-70% is accounted for by coding sequence . All 3 of the proteins predicted to be encoded by these genes display sequence differences compared to the human host homologues that may be of functional significance . These data bring the complement of cloned genes that encode activities in the pathway to seven, leaving only the gene encoding dihydroneopterin aldolase (DHNA) to be identified in the route from GTP to folate synthesis and folate turnover in the thymidylate cycle. Faraday Discuss, 2000, (116), 205 - 20; discussion 257-68 The effect of pH and ligand exchange on the redox properties of blue copper proteins; Canters GW et al.; A study of the structure and redox properties of the copper site in azurins by means of EXAFS, NMR, redox titrations, potentiometry, equilibrium cyclic voltammetry and rapid scan voltammetry on protein films is reported . The results are discussed in light of existing theories on structure and function of type-1 copper sites . The exit and entry of electrons take place through the C-terminal histidine ligand of the copper . The hydrophobic patch through which this residue penetrates the protein surface plays an important role in partner docking (cf . The rim of the porphyrin ring sticking through the surface of the cytochromes-c) . We find no experimental evidence for strain around the metal site . The active centre is able to maintain ET activity even in the presence of fairly gross disturbances of the site structure . The analysis of the thermodynamics of the redox reaction shows that the protein matrix and the solvent play an important role in 'tuning' the redox potential around a "design" value of around 300 mV at room temperature . The metal site appears "designed" to stabilise the Cu(II) instead of the Cu(I) form . The remarkable evolutionary success of the blue copper proteins is ascribed to the sturdy overall beta-sandwich structure of the protein in combination with a metal site that is structurally adaptable because three of its four ligands are located on a loop . The electronic "gate" that occurs in the middle of a hydrophobic patch allows for fine tuning of the docking patch for recognition purposes. Plant Mol Biol, 2000 Nov, 44(4), 513 - 27 Two rice MADS domain proteins interact with OsMADS1; Lim J et al.; OsMADS1 is a MADS box gene controlling flower development in rice . In order to learn more about the function of OsMADS1, we searched for cellular proteins interacting with OsMADS1 employing the yeast two-hybrid system . Two novel proteins with MADS domains, which were named OsMADS14 and OsMADS15, were isolated from a rice cDNA library . OsMADS14 and -15 are highly homologous to the maize MADS box gene ZAP1 which is an orthologue of the floral homeotic gene APETALA1 (AP1) . Interactions among the three MADS domain proteins were confirmed by in vitro experiments using GST-fused OsMADS1 expressed in Escherichia coli and in vitro translated proteins of OsMADS14 and -15 . We determined which domains in OsMADS1, -14, and -15 were required for protein-protein interaction employing the two-hybrid system and pull-down experiments . While the K domain was essential for protein-protein interaction, a region preceded by the K domain augmented this interaction . Interestingly, the C-terminal region of OsMADS1 functioned as a transcriptional activation domain in yeast and mammalian cells, while, on the other hand, the C domains of OsMADS14 and -15 exhibited only very weak transcriptional activator functionality, if any at all. Inorg Chem, 2000 Sep 18, 39(19), 4347 - 53 Synthetic models for the zinc sites in the methionine synthases; Chiou SJ et al.; The syntheses and molecular structures of a series of tetrahedral zinc complexes designed to model the active sites in Escherichia coli methionine synthases are reported . {PhTttBu}ZnBr (PhTttBu = phenyltris((tert-butylthio)-methyl)borate) was prepared and characterized crystallographically to provide entry into {S3}ZnX complexes . Metathesis with KSPh yielded the phenylthiolato complex, {PhTttBu}Zn(SPh), which represents a structural mimic of the homocysteine ligated form of the enzyme . Alternatively, {S2N}ZnX (X = Br, CH3, SPh) species were prepared using the new mixed-donor ligands, {Ph(pz)BttBu} (phenyl(pyrazolyl)bis((tert-butylthio)methyl)borate) and {Ph(pztBu)BttBu} (phenyl(3-tert-butylpyrazolyl)bis((tert- butylthio)methyl)borate) . Protonolysis of {Ph(pztBu)-BttBu}Zn(CH3) by PhSH in toluene yielded {Ph(pztBu)BttBu}Zn(SPh), a synthetic analogue of the homocysteine ligated form of cobalamin-independent methionine synthase (Met E) . The average Zn-S bond distance in {Ph-(pztBu)BttBu}Zn(SPh) of 2.37 A compares well with the EXAFS-derived distance of 2.31 A found in the homocysteine-bound form of Met E. Endocr Res, 2000 Nov, 26(4), 653 - 62 Regulation of arachidonic acid release in steroidogenesis: role of a new acyl-CoA thioestrase (ARTISt); Maloberti P et al.; It has been well established that arachidonic acid (AA) and its metabolism to leukotrienes plays an obligatory role in steroid production . The release of AA is regulated by hormone stimulation and protein phosphorylation . We have cloned a cDNA of a phosphoprotein with a molecular mass of 43 kDa (p43), purified from the cytosol of stimulated adrenal glands . This protein acts as intermediary in the stimulation of steroid synthesis through AA release, and has been found to be a member of a recently described acyl-CoA thioesterase family . In view of the mandatory role of this protein in the activation of AA-mediated steroidogenesis, the term Arachidonic acid-Related Thioesterase Involved in Steroidogenesis (ARTISt), is proposed for p43 . The present study describes the production of the recombinant protein by cDNA expression in Escherichia coli and its functional characterization . Recombinant acyl-CoA thioesterase was capable to release AA from the respective acyl-CoA, and this activity was affected by well-recognized inhibitors of AA release and metabolism: 4-bromophenacyl bromide (BPB) and nordihydroguariaretic acid (NDGA) . In addition, the inhibition of acyl-CoA thioesterase activity by NDGA correlates with the inhibition of steroid synthesis produced by this compound in adrenal cortex cells . Moreover, the recombinant protein was phosphorylated in vitro by PKA . These results provide the first evidence linking acyl-CoA thioesterases with the regulation of steroidogenesis, and support a regulatory role for acyl-CoA thioesterases in steroidogenic tissues, suggesting an alternative pathway for AA release in signal transduction. J Protein Chem, 2000 Aug, 19(6), 449 - 56 Chaperone-mediated refolding of recombinant prochymosin; Wei C et al.; It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable . Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present . The helping effect varies with the oxidation states of prochymosin . GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively . For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28% . Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI. Avian Dis, 2000 Oct-Dec, 44(4), 790 - 6 Nonspecific innate immunity against Escherichia coli infection in chickens induced by vaccine strains of Newcastle disease virus; Huang HJ et al.; The objective was to test the hypothesis that vaccine strains of Newcastle disease virus (NDV) induce nonspecific immunity against subsequent infection with Escherichia coli . White leghorn chickens at 5 wk of age were vaccinated with a NDV vaccine at various days before challenge exposure with O1:K1 strain of E . coli via an intra-air sac route . Immunity was determined on the basis of the viable number of E . coli in the spleen 24 hr after the infection . Roakin strain induced significant (P < 0.05) immunity against E . coli at 4, 6, and 8 days, and La Sota strain at 2, 4, and 8 days, postvaccination . Secondary NDV vaccination administered 14 days later failed to induce immunity against E . coli when chickens were infected 1 or 5 days after the vaccination . Significant (P < 0.05) suppression of this nonspecific immunity was observed in birds treated with corticosterone, 40 mg/kg in feed, given for three consecutive days immediately prior to the bacterial exposure but not in those treated prior to the period . The results indicate that innate immunity induced by the primary NDV vaccination may significantly suppress the multiplication of E . coli in chickens for a period of 2-8 days postvaccination . The NDV-induced immunity was inhibited by corticosterone, which is known to mediate physiological responses to stress. Avian Dis, 2000 Oct-Dec, 44(4), 759 - 69 Experimental Escherichia coli respiratory infection in broilers; Peighambari SM et al.; This study determined optimal conditions for experimental reproduction of colibacillosis by aerosol administration of avian pathogenic Escherichia coli to 2-to-4-wk-old broiler chickens . The basic model for reproducing disease was intranasal administration of approximately 10(4) mean embryo infectious dose of infectious bronchitis virus (IBV) followed by aerosol administration of an 02 or an 078 strain of E . coli in a Horsfall unit (100 ml of a suspension of 10(9) colony-forming units/ml over 40 min) . Scores were assigned to groups of infected chickens on the basis of deaths; frequency and severity of lesions in the air sacs, liver and heart; and recovery of the challenge E . coli 6 days post-E . coli infection . An interval of 4 days between the IBV and E . coli challenges was best whether the chickens received the IBV at 8 or 20 days of age . Typically, 50%-80% of the chickens developed airsacculitis and 0 to 29% of the chickens developed pericarditis or perihepatitis, with little or no mortality . Escherichia coli alone resulted in no deaths and 0 to 20% airsacculitis, but these percentages increased to 0 to 5% and 52%-60% when the E . coli aerosol was administered through a cone-shaped chamber . Administration of IBV alone failed to induce lesions . Recovery of the challenge E . coli from chickens did not correlate well with lesions . On the basis of these data, administration of IBV to 20-day-old chickens followed 4 days later by exposure to an avian pathogenic E . coli reproduces avian colibacillosis with the low mortality, high percentage of airsacculitis, and low percentage of septicemic lesions characteristic of the conditions seen in the natural disease. Mol Plant Microbe Interact, 2001 Jan, 14(1), 80 - 5 Inhibition of fungal appressorium formation by pepper (Capsicum annuum) esterase; Kim YS et al.; A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned . Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters . Inoculation of compatible unripe pepper fruits with C . gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit . The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner . An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea . Inhibition of appressorium formation in M . grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol . The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus . Taken together, the PepEST esterase activity can inhibit appressorium formation of C . gloeosporioides, which may result in protection of the unripe fruit against the fungus. Mol Plant Microbe Interact, 2001 Jan, 14(1), 63 - 71 Functional screening yields a new beta-1,4-endoglucanase gene from Heterodera glycines that may be the product of recent gene duplication; Yan Y et al.; Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate . Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a beta-1,4-endoglucanase gene (HG-eng-2), previously isolated from H . glycines . A subgroup of identical "HG-eng-2-like" clones had considerable differences in the 5' untranslated region compared with HG-eng-2 and were designated HG-eng-3 . One H . glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H . glycines . The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5' flanking regions . The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion. Ann N Y Acad Sci, 2000, 921, 165 - 74 Regulation of vasoactive intestinal peptide receptor expression in developing nervous systems; Karacay B et al.; Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide that has several functions, including the regulation of water and electrolyte secretion, hormone and cytokine release, bronchodilitation, and neurogenesis . VIP effects are mediated by specific G-protein coupled receptors . Three distinct receptor subtypes, with differing affinity for VIP, have been cloned and characterized as receptors 1 and 2 (VPAC1 and VPAC2) and pituitary adenylate cyclase activating polypeptide receptor (PAC1) . Our laboratory has demonstrated that upregulation of VPAC1 in SK-N-SH neuroblastoma cells results in marked shift in cell type to the glial lineage with a corresponding loss of neuronal lineage and suppression of xenograft tumor growth . To understand the molecular mechanisms responsible for regulation of the VPAC1 gene in neuronal lineage, we have cloned and sequenced 2.6-kb of the 5'-flanking sequences of the human VPAC1 gene . Sequence analysis demonstrated that the human VPAC1 promoter sequence contains putative binding sites for several known transcription factors, including Sp1, NFkB, and cETS-1 . To study the temporal and spatial expression pattern of human VPAC1 promoter sequences, we have generated transgenic mice expressing the bacterial beta-galactosidase gene under the control of the 2.6-kb 5'-flanking and promoter sequence of the human VPAC1 gene . Transgene expression was detected in brain, spinal cord, and lung in 14-day-old animals . Taken together, these results demonstrate that VPAC1 may play an important role in the nervous system, and suggest a role for VIP in neuronal differentiation. Ann N Y Acad Sci, 2000, 921, 157 - 64 Molecular characterization of the VIP receptor transcriptional repressor protein; Pei L; The rat type 1 VIP receptor transcriptional repressor protein (VIPR-RP) is a recently isolated novel transcription factor . In the study reported here, the functional domains of VIPR-RP were characterized . To map the DNA binding domain, various regions of VIPR-RP were either transcribed and translated in vitro or expressed in and purified from E . Coli as a glutathione S-transferase (GST) fusion . The ability of the truncated proteins to bind to VIPR-RP specific binding sequence was tested by gel mobility shift assays . The results indicated that the amino acid sequences between 367 and 475 play an essential role for VIPR-RP DNA binding . To determine the amino acid sequences required for transcriptional repression, fusion proteins containing the GAL4 DNA binding domain and various parts of VIPR-RP were constructed, and their ability to repress transcription of the reporter gene containing GAL4 DNA binding sequences were tested in transiently transfected COS7 cells . The results showed that VIPR-RP contains two separate transcriptional repression domains located between amino acids 50 to 101 and 470 to 527. Virchows Arch, 2000 Dec, 437(6), 656 - 61 Phlegmonous colitis: a specific and severe complication of chronic hepatic disease; Satoh T et al.; Phlegmonous colitis (PC) is an acute infectious entity caused by bacteria . In this study, we reviewed 8,822 autopsy cases and found 13 cases of PC (0.15%) . PC affected 2.43% of patients with hepatic cirrhosis or subacute liver atrophy, both of which were considered to be due to hepatitis viral infection . Before autopsy, none of the cases studied was suspected to involve PC, irrespective of the immediate cause of patient death . Thirteen autopsy cases showed some or all of the following pathohistologic characteristics: (1) involvement of the cecum (9 cases, 76.9%), (2) phlegmonous inflammatory changes and edema in the submucosa (100%), (3) bacterial infection (100%), (4) no microscopically detectable mucosal injuries (12 cases, 92.3%), and (5) acute serositis (peritonitis) (2 cases, 15.4%) . These results suggest that PC is an unrecognized, but fatal complication of patients with some hepatic diseases and that PC has pathohistologic characteristics in common with previously reported spontaneous bacterial peritonitis in animal models . PC probably arises due to spontaneous infection in patients with hepatic cirrhosis. Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2512 - 6 Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp . strain O-7; Tsujibo H et al.; The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme . The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa . A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane . The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases. Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2445 - 53 Family 19 chitinases from Streptomyces thermoviolaceus OPC-520: molecular cloning and characterization; Tsujibo H et al.; Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced . The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively . Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain . Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases . The cloned Chi35 and Chi25 were purified from E . coli and S . lividans as a host, respectively . The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70 degrees C, respectively . Chi35 bound to chitin, Avicel, and xylan . On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35 . These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain . Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25 . In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain . However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains. Yakugaku Zasshi, 2000 Dec, 120(12), 1347 - 57 {Roles of human cytochrome P450 enzymes involved in drug metabolism and toxicological studies}; Yamazaki H; Multiple forms of cytochrome P450 (P450 or CYP) enzymes play important roles in the oxidation of structurally diverse xenobiotics and endobiotics . Interindividual variations in the level and activity of P450 enzymes were investigated in the human liver microsomes . Although the total P450 content was higher in Caucasian samples than in Japanese ones, the relative levels (percentage of total P450) of individual forms of P450 determined immunochemically were not very different . CYP3A (about 30% of total P450) and CYP2C (about 20%) enzymes were major forms . Different P450 enzymes in the human liver play major roles in a variety of drug oxidations and the hepatic contents of these P450 forms could be affective to determine which P450 enzymes play major roles in drug metabolism in individual humans . Recently recombinant P450 enzymes from different sources, e.g., microsomes of human lymphoblastoid cells, of yeast, and insect cells infected with baculovirus systems, and Escherichia coli membranes containing coexpressed P450 and reductase, have been widely used for drug metabolism research . However, the marker activities or kinetic parameters of human P450 enzymes reported are not always similar . Cytochrome b5 can enhance the activities of recombinant P450 systems in some cases using different mechanisms . These differences in activities may be a critical factor for understanding the roles of human P450 enzymes involved in drug metabolism . This review provides useful information for the study of drug biotransformation in humans and for the basis of drug toxicities and carcinogenesis. Hinyokika Kiyo, 2000 Nov, 46(11), 803 - 5 {A case of effective endotoxin adsorption therapy for septic shock due to acute pyelonephritis}; Abe K et al.; Although septic shock has a high mortality rate of 43%, recently the endotoxin adsorption column was established and its efficacy is interesting . We report a very effective case of endotoxin adsorption the rapy for septic shock due to acute pyelonephritis . A 59-year-old man with chief complaints of pyrexia and right backache was referred to our hospital with a small right ureteral stone (4 mm) associated with a low degree of right hydronephrosis . Since it was diagnosed as right acute pyelonephritis, antibiotics were administered; and then septic shock occurred on the day of hospitalization . Endotoxin adsorption therapy was performed for two days and hemodynamic stability was achieved . The concentration of blood endotoxin was reduced remarkably and the efficacy of endotoxin adsorption therapy was suggested. Intensive Care Med, 2000 Nov, 26(11), 1670 - 80 Hemodynamic, biochemical and morphological changes induced by aminoguanidine in normal and septic sheep; Lorente JA et al.; OBJECTIVE: To define the acute hemodynamic, metabolic, and morphological changes induced by aminoguanidine, a selective iNOS inhibitor, in septic sheep . DESIGN: Prospective, nonrandomized animal study . SETTING: Animal research facility in a University Hospital . INTERVENTIONS: Adult sheep, sedated and mechanically ventilated, were monitored with a pulmonary arterial catheter and an ultrasonic blood flow probe in the mesenteric artery, to measure the systemic (Q(TOT)I) and the mesenteric (Q(MES)I) blood flow indices, and an ileal tonometer . Four groups of sheep were studied: nonseptic, septic, nonseptic treated with aminoguanidine, and septic treated with aminoguanidine (100 mg kg(-1) h(-1)) (n = 6 for each group) . Sepsis was induced by the intravenous administration of E . coli . Hemodynamic and biochemical parameters were measured during 300 min . Histological changes in the liver and small intestinal mucosa were analyzed at the end of the experiment . MEASUREMENTS AND MAIN RESULTS: In nonseptic animals, aminoguanidine slightly increased mean systemic arterial pressure (MAP), decreased Q(TOT)I, and increased vascular resistance index (SVRI) and pulmonary vascular resistance index . Q(MEs)I did not change and Q(MES)I/Q(ToT)I increased . Aminoguanidine also induced intestinal intramucosal hypercarbia, hyperlactatemia, acidemia, hypoglycemia, and morphological signs indicative of tissue ischemia in the small intestinal mucosa . In septic sheep, aminoguanidine increased SVRI and MAP only at 4 h after the septic challenge and thereafter, and worsened gas exchange . CONCLUSIONS: In this model, exogenous administration of aminoguanidine induces beneficial hemodynamic effects 4 h after the septic challenge . In normal animals, however, aminoguanidine was associated with hypoglycemia, acidosis, hyperlactatemia, and intestinal mucosal ischemia. Analyst, 2000 Nov, 125(11), 1924 - 7 Enzymatic activity measurement of phospholipid hydroperoxide glutathione peroxidase by capillary electrophoresis; Ru QH et al.; Based on the separation of 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OOH) and 1-palmitoyl-2-(13-hydroxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OH) and the quantitative determination of PC-OH, the enzymatic activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) can be measured by capillary electrophoresis . The separation was carried out in a fused-silica capillary (30 cm x 100 microns id) at 15 kV positive voltage . Sodium borate (100 mM; pH = 8.4) was used as the running buffer, and the photodiode array detector wavelength was 232 nm . The determination can be completed in 5 min . The detection limit was 5 pmol; and the relative standard deviation (RSD) of the peak area was less than 1% with an average recovery of 98.6% . Compared with traditional methods such as HPLC and spectrophotometry, it is faster and more convenient . Using capillary electrophoresis, the enzymatic activities of PHGPx expressed by the rice PHGPx gene in E . coli . M15 was determined as 1.25 x 10(-5) mumol min-1, and the specific activity of partially purified trans-gene PHGPx was 3.1 x 10(-2) mumol min-1 per mg . The stability of the trans-gene PHGPx was also studied. Nucl Med Commun, 2000 Nov, 21(11), 1043 - 50 A 99Tcm-labelled leukotriene B4 receptor antagonist for scintigraphic detection of infection in rabbits; Brouwers AH et al.; In a search for a rapid and accurate imaging agent for scintigraphic detection of infection and inflammation, an LTB4 receptor antagonist, 99Tcm-RP517, which contains the hydrazino nicotinamide moiety, has been developed recently . To study the in vivo behaviour of 99Tcm-RP517, rabbits with Escherichia coli infection were injected intravenously with 99Tcm-RP517 . Gamma camera images were obtained and ex vivo bio-distribution was determined at several hours post-injection (p.i.) . In a separate set of rabbits the choledochal duct was cannulated to quantitatively monitor the hepatobiliary clearance of the radiopharmaceutical . The receptor binding fraction of the radiolabelled RP517 exceeded 70% . Accumulation of 99Tcm-RP517 in the abscess was visualized as early as 1 h p.i . Due to rapid blood clearance (t1/2 alpha=18+/-0.6 min, t1/2 beta=6.5+/-0.4 h) and high abscess uptake, the abscess-to-muscle ratios increased with time from 7.0+/-2.3 at 1 h p.i . to 44.3+/-4.6 at 20 h p.i . The agent mainly cleared via the hepatobiliary route: 50% of the radiolabel was recovered in the small bowel at 1 h p.i., whereas 85% was found in cecum and sigmoid at 20 h p.i . In conclusion, 99Tcm-RP517 rapidly visualized E . coli abscesses in rabbits . The agent rapidly cleared from the blood, mainly via the hepatobiliary route . High abscess-to-background ratios were achieved . The accumulation in the intestines could limit the applicability of this agent for detecting infectious processes in the abdominal area . The development of a more hydrophilic analogue of 99Tcm-RP517 could improve the clinical applicability of this agent. Respirology, 2000 Dec, 5(4), 389 - 92 Use of montelukast in the treatment of early childhood wheezing from clinical experience with three cases; Ng DK et al.; Leukotrienes were found to be raised in respiratory syncytial virus bronchiolitis . Montelukast is a cysteinyl leukotrienes antagonist . We report our experience with the use of montelukast in three young children from 5-months to 20-months old . The first case was a 5-month-old boy with previous good health . He had prolonged respiratory distress secondary to adenovirus type 3 infection . The second case was a 20-month-old boy with bronchopulmonary dysplasia . He had respiratory syncytial virus and an adenovirus type 3 infection leading to prolonged wheeze . The third case was a 20-month-old girl with chronic lung disorder after an episode of severe E . coli pneumonia at 1 month old . She developed acute virus-negative severe wheeze after a few days of running nose and low-grade fever . All three cases responded poorly to inhaled steroids and bronchodilators . Addition of montelukast was associated with marked clinical improvement within 1 week . The three cases were very heterogeneous and differed from usual simple virus-induced acute bronchiolitis . The use of multiple drugs including montelukast did not enable any definite conclusions; however, the addition of montelukast was closely related to clinical improvement . Further studies in the use of montelukast in severe virus-induced bronchiolitis are warranted. Pediatr Int, 2000 Dec, 42(6), 637 - 41 Plasma levels of granulocyte elastase-alpha1-proteinase inhibitor complex in children with hemolytic uremic syndrome caused by verotoxin-producing Escherichia coli; Ishikawa N et al.; BACKGROUND: Activated neutrophils play an important role in the pathogenesis of renal injury in humans and in experimental models of hemolytic uremic syndrome (HUS) . To evaluate the clinical significance of the circulating granulocyte elastase-alpha1-proteinase inhibitor complex (GEPIC), which is a marker of neutrophil activation, we investigated the plasma concentrations of GEPIC in children with hemolytic uremic syndrome (HUS) associated with verotoxin-producing Escherichia coli (VTEC), VTEC gastroenteritis without HUS and in normal controls . METHODS: Of 22 children (1-19 years of age; mean age 5.5 years) with VTEC infection, nine were diagnosed with HUS . Plasma GEPIC, soluble thrombomodulin (sTM) and thrombin-antithrombin-III complex (TAT) levels were measured by ELISA . RESULTS: The number of polymorphonuclear leukocytes and the levels of plasma GEPIC in patients with HUS were significantly higher than those in non-HUS (9850+/-5091 vs . 5278+/-3327 /microL, P<0.05; 432.1+/-211.7 vs . 188.3+/-117.0 ng/mL, P<0.01) or control subjects (9850+/-5091 vs . 4728+/-1977 /microL, P<0.05; 432.1+/-211.7 vs . 105.9+/-51.1 ng/mL, P<0.001) . Furthermore, plasma GEPIC levels showed a positive correlation with sTM (r = 0.522; P<0.01), a marker of endothelial cell injury, and TAT (r = 0.594; P<0.01), a marker of thrombin activity . CONCLUSIONS: These results suggest that an increase in circulating GEPIC levels in patients with VTEC-associated HUS may be related to endothelial injury, which may possibly lead to a more severe episode of this disease. Yi Chuan Xue Bao, 2000, 27(10), 925 - 31 {Subcloning and sequencing of DNA fragment related to salt tolerance in Sinorhizobium fredii RT19}; Bian XL et al.; A 23 kb DNA fragment related to salt tolerance was obtained from the gene library of S . fredii strain RT19 . In this study, BamH I was selected to digest 23 kb DNA fragment into different length of DNA fragments . The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E . coli S17-1 on selective medium and three transformants TR were obtained . Two-parental mating experiments were carried out with these transformants as donor and salt sensitive S . fredii strain RC3-3 as recipient, and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4 mol/L NaCl . Thus, a 4.4 kb DNA fragment related to salt tolerance was obtained . Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing . Subsequently, sequencing and analysis of 4.4 kb DNA fragment showed that fixO, fixN genes and three ORFs were obtained. Yi Chuan Xue Bao, 2000, 27(10), 911 - 7 {Properties and nucleotide sequence of mitochondrial plasmid-like DNA pC1 of cucumber}; Li JY et al.; Four kinds of plasmid-like DNA, designated pC1, pC2, pC3 and pC4 were found in the mitochondrion of cucumber Jinyan No . 4 . The circle plasmid-like DNA pC1 was cloned into the EcoR I site of pUC19 using E . coli JM109 as host . The cloned pC1 DNA was isolated and used as probes in Southern analyses of total mitochondrial DNA, nuclear DNA and chloroplast DNA . Evidences were obtained that the pC1 did not show any homology with nuclear, chloroplast, main mitochondrial genomes and the other plasmid-like DNAs . Sequence analysis revealed that pC1 was 2,889 bp long . It contained many forward and reverse repeat sequences . Three main open reading frames in pC1 were longer than 800 bp . Computer-assisted searching the nucleotide sequence in GenBank database revealed pC1 had no significant homology with known sequences of mitochondrial and plasmid-like DNA, but had homology with the E . coli, Mycobaterium tuberculosis and Anacystis nidulans genomes . The predicted proteins of pC1 main ORFs show homology with the sulfate transport system in bacteria, alga and liverwort . It suggested that pC1 may encode functional proteins. Viral Immunol, 2000, 13(4), 533 - 45 An immunodominant, cross-reactive B-cell epitope region is located at the C-terminal part of the hamster polyomavirus major capsid protein VP1; Siray H et al.; The VP1 represents the major capsid protein of the hamster polyomavirus (HaPV) . Here we describe the mapping of epitopes along the VP1 using Escherichia coli-expressed VP1-dihydrofolate reductase (DHFR) fusion proteins and PepScan analysis . By use of DHFR fusion proteins an immunodominant region was localized in the C-terminal part of VP1 between amino acids 320-384 . Further epitopes are located in the regions amino acids 1-133 and amino acids 133-320, respectively . There were no obvious differences in the reactivity between sera of tumor-bearing and papilloma-free naturally HaPV-infected hamsters . In contrast, PepScan analysis revealed linear epitopes in the regions amino acids 79-97 and amino acids 353-367 for tumor-bearing animals and amino acids 101-113 and amino acids 165-179 for papilloma-free animals . The region between amino acids 320-384 of HaPV-VP1 was found to be involved in cross-reactivity of VP1 from HaPV and other polyomaviruses . Previously we have demonstrated that heterologous expression of HaPV-VP1 allowed the formation of virus-like particles (VLPs) . From epitope mapping data and structural predictions it has been suggested that HaPV-VP1-VLPs may tolerate foreign peptides in the region amino acids 81-88 and the C-terminal part of VP1. Res Microbiol, 2000 Dec, 151(10), 865 - 71 Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats; Goffaux F et al.; Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats . They produce typical histological lesions called 'attaching and effacing' lesions . Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection . The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates . A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates . The LEE was inserted in the selC region in only 12% of the isolates . The eae, tir, espA and espB genes were analyzed by multiplex PCR . The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%) . Moreover, the espD gene was also present in dog and cat EPEC . The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 599 - 602 {Isolation and characterization of promoters from Phanerochaete chrysosporium}; Li W et al.; Promoter-probe vector pSUPV8 was used to clone promoters from Phanerochaete chrysosporium directly in Escherichia coli . Six hygromycin B-resistant recombinants were obtained and two inserted fragments of them were sequenced . The results indicated that they contain several sequences similar to eukaryotic cis regulatory elements . Only pCH6 could transform P . chrysosporium into hygromecin B resistance . PCR and dot hybridization analysis indicated that it was introduced into P . chrysosporium successfully and leaded to the HmB-resistance . The results demonstrated that HmB-resistant gene (hph) could be used as an ideal reporter gene for P . chrysosporium transformation. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 595 - 8 {Expression of 2.1 kb enhancin gene fragment from Helicoverpa armigera granulosis virus in Escherichia coli}; Ou Y et al.; The 2.1 kb fragment of enhancin gene from Helicoverpa armigera granulosis virus was inserted into vector pQE-30 and expressed successfully in E . coli M15(pREP4) . The synergy of expression product(P78) on AcMNPV against the larvae of Plutella xylostella was also studied . The results indicated that the percentage of correct mortality of the larvae increased 27.88%-32.92% in 10 post-infection days. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 578 - 81 {Construction and application of an Escherichia coli high effective expression vector with an enhancer}; Luo WX et al.; In this study, we constructed a high effective fusion expression-vector in E . coli . This vector, pTO-T7, was characterized as: (1) an enhancer from tobacco mosaic virus (TMV), omega sequence, was ligated in front of a T7 promoter in the regulatory sequence; (2) the multi-cloning sites include eight restriction enzyme sites . It can facilitate fusion or nonfusion expression; (3) the N terminal of a fusion protein starts with the first 12 amino acids of T7 gene 10, and the C terminal is the hexahistidine tag; (4) kanmycin resistance gene was used as a selective marker . EGFP gene was inserted into pTO-T7 vector as a reporter gene . Expression data showed that fused-EGFP accounted to more than 50% of the total E . coli protein, and more than 90% of which was soluble . The fluorescence characters of fused-EGFP were also studied . The expression yield of target gene from plasmid pTO-T7 compared with that from pT-T7 without omega sequence suggested that omega sequence in pTO-T7 can improve the expression of target gene significantly. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 557 - 60 {Gene cloning, expression and purification of its production of recombinant human superoxide dismutase}; Zhang Y et al.; Human SOD cDNA was cloned and constructed an expression plasmid with high sufficient and stabilility expression in E . coli . The rhSOD cDNA was amplified by RT-PCR with the template of the total RNA extracted from human liver tissue . The expression plasmid, pLY-4/rhSOD, containing rhSOD cDNA, was transformed into the E . coli JF1125 . The sequence of the cloned rhSOD cDNA was identified with the reported data . The expression level reached to more than 68% of total bacteria proteins; The technology for protein renature and purification was efficiency and fast . The purity of the final products reached more than 98% . The value of bioactivity was determined as 2529 u/mg . This study gave enough support for production of rhSOD by biotechnology. Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 551 - 6 {Studies on the construction and expression of recombinant human fusion gene Nm23-H1/HbFGF in Escherichia coli}; Sun YM et al.; First, a piece of intermediate nucleic acid chain was designed according to the nucleic acid sequences of Nm23-H1 and HbFGF cDNA, then it was combined with the upstream primer of Nm23-H1 or the downstream primer of HbFGF to perform Polymerase Chain Reaction (PCR) respectively . The fusion gene Nm23-H1/HbFGF was constructed by four steps of PCR, and it was cloned into the plasmid vector pBV220 . The recombinant was induced at 42 degrees C and analyzed by SDS-PAGE . Results show that the fusion gene Nm23-H1/HbFGF highly expresses its product in inclusion body in E . coli BL21(DE3) . The expressing product is 14% of the total bacterial protein and it is 34 kD . ELISA assay and Western blot indicate that the inclusion body contains antigens of Nm23-H1 and HbFGF . By denaturation, renaturation and purification, the inclusion body was purified . Determination and biological activity assay show that the purified product contains two kinds of antigens which have biological activities of Nm23-H1 and HbFGF . All these data establish good base to study the tumor suppressor activity and oncogenicity of the fusion gene Nm23-H1/HbFGF in eukaryocyte. Genetika, 2000 Dec, 36(12), 1614 - 21 {Localization of proteins, immunologically similar to Eschericha coli RecA protein, in structures of cultured cells of HeLa and K-562 cell lines}; Loseva EF et al.; Earlier, polyclonal antibodies to the Escherichia coli RecA protein revealed immunologically related proteins only in the reproductive tissues of various eukaryotes and the proteins were localized to the various structures of the synaptonemal complex (SC) . Indirect immunofluorescent FITC staining with polyclonal antibodies against RecA detected RecA-like antigens in the cell center (centrosome) and the replicated centrioles of fixed HeLa and K-562 cells . Human and mouse genes, which were previously cloned by immunological relatedness of their products to E . coli RecA, were used as hybridization probes in Northern blot analysis of the transcription activity of similar genes in K-562 cells . A gene homologous to the cloned mouse gene proved transcribed in the cells studied. Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 463 - 8 {Preliminary studies on outer membrane protein (OMP) patterns of Escherichia coli O1, O2 and O78 isolates of chicken origin}; Gao S et al.; Omp patterns of twenty-five isolates of Escherichia coli serotype O1, O2 and O78 of chicken origin from Jiangsu area were determined . The outer membrane proteins of these isolates were isolated with the improved N-lauroylsarcosine method and analyzed by SDS-PAGE . Major outer membrane proteins bands contributed for OMP patterns were visualized with Coomassie brilliant blue staining . Two OMP patterns were divided in eight of O1 isolates and nine O2 isolates respectively . Among these OMP patterns, 1 OMP pattern was shared by these 2 different O serotypes isolates, and eight of O78 isolates also shared this OMP pattern . These results indicated that the OMP patterns of avian pathogenic Escherichia coli isolates of O1, O2 and O78 from this area were heterogeneous, and the same OMP patterns was presented in these 3 serotypes. Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 455 - 62 {DNA polymorphism in the genomes of different Escherichia coli strains}; Wang Y et al.; Bacteria have been traditionally classified on the basis of their morphology, biochemical reaction, serology and etc . However, in some case these methods could not authenticate the closely related bacteria strains . In this study, we cloned two repetitive DNA sequences with 0.9 and 0.6 kb in length from Escherichia coli K12 strain JM109 and designated as ECR-1 and ECR-6 respectively . Using ECR-1 and ECR-6 sequences or their combination as the probes for DNA polymorphism analysis, we were be able to develop a molecular method of biotyping for the identification of very closely related strains of Escherichia coli . Both of ECR-1 and ECR-6 probes could be applied for the taxonomy, epidemiological and microecological studies, and clinical diagnosis for pathogenic Escherichia coli strains. Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 438 - 42 {Subcloning of copper resistance promoters from Escherichia coli}; Liu Z et al.; There were two promoters, PpcoA and PpcoE, in the copper resistant determinant from Escherichia coli . Either of them contains the copper box . In order to confirm the importance of copper box in the copper resistance promoters and to study their characteristics, several fragments of both promoters were subcloned using pUCD615 as reporter vector into its SmaI site . The results of restriction endonuclease digestion and the results of reporter gene lux-luciferase activities indicated that both of the Ppco short-lux fusions didn't show any luciferase activities, which indicated that these two fragments (Ppco short) couldn't act as promoters, and thus confirming the copper box was essential to copper resistance . If there were no copper box in the Ppco promoters, there would be no copper resistance in the E . coli. Wei Sheng Wu Xue Bao, 1997 Oct, 37(5), 344 - 8 {Cloning, expression and purification of the chaperonin GroESL in Escherichia coli}; Zhou Y et al.; The DNA fragment encoding the molecular chaperion GroESL was subcloned into high-expression vector pKC220, and the GroESL were high expressed in the E . coli strain harboring the recombinant plasmid by high temperature induction . The amount of the expressed GroEL and GroES protein were about 40% and 15% of the total cellular proteins, respectively . These two subunits were both purified from E . coli by (NH4)2SO4 salt out DEAE-52 chromography and Sephadex G-50 chromography. Auton Neurosci, 2000 Dec 20, 85(1-3), 78 - 82 Multiple neural mechanisms of fever; Szekely M et al.; In rats, fevers induced by moderate-to-high doses of intravenous lipopolysaccharide consist of three phases (phases 1, 2 and 3) with body temperature peaks at approximately 1, 2, and 5 h postinjection, respectively . In this study, the effects of bilateral truncal subdiaphragmatic vagotomy and intraperitoneal capsaicin desensitization on febrile phases 1-3 were assessed in adult Wistar rats . Surgical vagotomy was performed approximately 30 d before the experiment; this procedure interrupts both afferent and efferent vagal fibers . Capsaicin was administered intraperitoneally in two consecutive injections (2 and 3 mg/kg, 3 h apart) 1 week prior to the experiment; this procedure desensitizes afferent fibers, primarily within the abdominal cavity, and does not lead to the known thermal effects of systemic capsaicin desensitization . At a neutral ambient temperature, the rats were given Escherichia coli lipopolysaccharide (10 microg/kg) through a preimplanted jugular catheter, and their colonic temperature wes measured by thermocouples for 7 h . The control rats exhibited the typical triphasic febrile responses . Confirming our earlier studies, subdiaphragmatic vagotomy did not affect phases 1 and 2; it did, however, result in a 2.5-fold reduction of phase 3 . Capsaicin desensitization modified the febrile response differently: phases 2 and 3 were unaffected, but phase 1 disappeared . We suggest that neural afferent fibers (nonvagal but perhaps vagal as well) play an important role in the early febrile response (phase 1) by transducing peripheral pyrogenic signals to the brain . We also suggest that vagal efferent fibers are likely to participate in the later febrile response (phase 3) via an unknown mechanism. Auton Neurosci, 2000 Dec 20, 85(1-3), 133 - 40 Lipopolysaccharide transport from the peritoneal cavity to the blood: is it controlled by the vagus nerve? Romanovsky AA, Ivanov AI, Lenczowski MJ, Kulchitsky VA, Van Dam AM, Poole S, Homer LD, Tilders FJ. Vagotomy suppresses fever and hyperalgesia caused by intraperitoneal lipopolysaccharide (LPS) but has little effect on the febrile response to intravenous or intramuscular LPS . This suggests that some vagus-mediated mechanisms are recruited only when LPS is administered via the intraperitoneal route . We hypothesized that such mechanisms are associated with LPS transport from the peritoneal cavity to the circulation . Adult Wistar rats underwent total subdiaphragmatic, bilateral selective celiac, or sham vagotomy . On day 28-32 after surgery, they were injected IP with Escherichia coli LPS (5, 20, or 100 microg/kg) or saline and decapitated 90 min thereafter . Their plasma levels of LPS and their plasma interleukin-6, adrenocorticotropin, and corticosterone responses to LPS were measured . Success of intraperitoneal administration of LPS was verified by increased interleukin-1beta and interleukin-6 concentrations in the peritoneal lavage fluid . Effectiveness of vagotomies was confirmed by increased stomach mass (food retention) and pancreas mass (hypertrophy) . In the shams, LPS caused a dose-dependent endotoxemia and increased plasma levels of interleukin-6, adrenocorticotropin, and corticosterone . Neither celiac nor total vagotomy affected any of these responses . LPS escapes from the peritoneal cavity by two primary routes, viz., the hematogenous (via the portal vein) and lymphogenous (via the lymphatic system) . The design of the present study did not allow for evaluating the rapid, hematogenous transport . The results obtained suggest that the abdominal vagus does not control the slow . lymphogenous escape of LPS from the peritoneal cavity. Auton Neurosci, 2000 Dec 20, 85(1-3), 111 - 8 Does the formation of lipopolysaccharide tolerance require intact vagal innervation of the liver? Ivanov AI, Kulchitsky VA, Sugimoto N, Simons CT, Romanovsky AA. The study was designed to test whether intact vagal innervation of the liver is required for the formation of tolerance to lipopolysaccharide (LPS) . Wistar rats were subjected to either denervation of the liver (transection of the hepatic and both celiac branches of the abdominal vagus) or sham surgery . Two weeks later, each rat had an osmotic pump implanted subcutaneously . The pump was filled with either a suspension of Escherichia coli LPS (18 mg/ml) in saline or saline alone . Via a catheter, the pump delivered its content into the right jugular vein at a rate of approximately 0.72 microl/kg/h (approximately 13 microg/kg/h of LPS) over 28 d . On day 25 of the infusion, each animal had another catheter implanted into the left jugular vein . Three days later, each rat was injected with a lethal bolus dose of LPS (15 mg/kg) and had its colonic temperature recorded . The saline-infused sham-operated rats responded to the bolus injection of LPS with hypothermia followed by a fever (mean response magnitude 1.0+/-0.2 degrees C); 91% of the animals died within 24 h . The LPS-primed shams developed marked tolerance: When challenged with a lethal dose of LPS, they exhibited a significantly smaller thermal response (magnitude 0.5 +/- 0.2 degrees C) and none died . No group of the vagotomized animals, whether LPS- or saline-primed, became tolerant: Both groups exhibited similar hypothermic responses to the bolus LPS injection and a substantial mortality rate (40 and 100%, respectively) . The study shows that prolonged infusion of low doses of LPS leads to the formation of tolerance and that vagal denervation of the liver by hepato-celiac vagotomy suppresses this process . The mechanisms of vagal control of the formation of LPS tolerance remain speculative. Structure Fold Des, 2000 Dec 15, 8(12), R237 - 41 DNA mismatch repair: the hands of a genome guardian; Hopfner KP et al.; DNA mismatch repair (MMR) is initiated when the MutS protein recognizes damaged DNA . Crystal structures of MutS bound to mispaired and unpaired DNA show how MutS distinguishes damaged from undamaged DNA and explain how a broad variety of DNA mismatch lesions can be detected . The structures suggest mechanisms for the ATP-induced structural regulation of multistep DNA repair processes. Structure Fold Des, 2000 Dec 15, 8(12), 1319 - 28 Dramatic structural and thermodynamic consequences of repacking a protein's hydrophobic core; Willis MA et al.; BACKGROUND: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies . A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized . RESULTS: We present a structural and thermodynamic analysis of Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core . The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface . The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop . Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding . CONCLUSIONS: The structure of Ala2Ile2-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold . Structural disorder at the N and C termini of Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop. Nippon Yakurigaku Zasshi, 2000 Dec, 116(6), 359 - 70 {Simplified method for gene transfer and expression by recombinant adenoviruses}; Maruyama Y et al.; Recombinant adenoviruses are attracting a great deal of attention as a highly efficient gene delivery technology and used for in vitro and in vivo gene expression in science . However, among traditional methods, there have been many difficulties and no simple procedure to generate recombinant adenoviruses . Since almost of all these methods involve the process of homologous recombination in a mammalian packaging cell line, the problems are low efficiency of homologous recombination, the need for complicated techniques and the demand for a long time to generate recombinant viruses . These problems have prevented widespread use of adenovirus technology as effective gene transfer tools . In the last few years, there have been several significant and innovative advances in adenoviral technologies, which include a new generation of vectors, the ease of vector manipulation and improvement of the viral production system with homologous recombination in E . coli . Here, we describe the easier and more efficient viral production systems and provide a practical manual for generating recombinant adenoviruses based on one of such systems. Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1488 - 91 Purification, crystallization and preliminary X-ray crystallographic analysis of ATP-phosphoribosyltransferase from Escherichia coli; Lohkamp B et al.; ATP-phosphoribosyltransferase (ATP-PRT) from Escherichia coli has been purified and crystals were obtained by the vapour-diffusion method using sodium tartrate as a precipitant . Dynamic light scattering was used to assess conditions for the monodispersity of the enzyme . The crystals are trigonal, space group R32, with unit-cell parameters a = b = 133.6, c= 114.1 A (at 100 K), and diffract to 2.7 A on a synchrotron X-ray source . The asymmetric unit is likely to contain one molecule, corresponding to a packing density of 2.9 A3 Da(-1) . A model for the quaternary structure is proposed based on the crystallographic symmetry. Arch Biochem Biophys, 2000 Nov 15, 383(2), 309 - 17 The juxtamembrane region of the epidermal growth factor receptor is required for phosphorylation of Galpha(s); Poppleton HM et al.; We have previously demonstrated that Galpha(s) associates with the juxtamembrane region of the epidermal growth factor (EGF) receptor (EGFR) and that the EGFR can phosphorylate and activate this G protein (H . Poppleton et al., 1996, J . Biol . Chem . 271, 6947-6951; H . Sun et al., 1995, Proc . Natl . Acad . Sci . USA 92, 2229-2233) . In this report, we have employed peptides EGFR-13 and EGFR-14 (corresponding to amino acids 645-657 and 679-692 in the EGFR, respectively) which disrupt the association of Galpha(s) with the EGFR to investigate whether or not this region of the EGFR is required for phosphorylation of the G protein . EGFR-13 increased the tyrosine phosphorylation of G(alpha)s by two-fold whereas EGFR-14 decreased the phosphorylation of the G protein . Phosphorylation of EGFR-13 on the threonine residue corresponding to Thr654 of the EGFR obliterated the ability of the peptide to increase Galpha(s) phosphorylation . EGFR-13 and EGFR-14, but not phospho-EGFR-13, competed for the association of the EGFR with Galpha(s) . A peptide betaIII-2 corresponding to amino acids Arg259-Lys273 in the beta2-adrenergic receptor which competes for association of Galpha(s) with the EGFR and increases protein tyrosine kinase activity of the EGFR could mimic the effects of EGFR-13 . Among the three peptides (EGFR-13, EGFR-14, and betaIII-2) that interfere with association of Galpha(s) to the EGFR, only EGFR-13 and betaIII-2 have been shown to activate the G protein . Polylysine which increases EGFR tyrosine kinase activity but does not interfere with association of Galpha(s) and EGFR also augmented phosphorylation of Galpha(s) by the EGFR . Phosphopeptide mapping demonstrated that EGFR-13 and polylysine increased phosphorylation of Galpha(s) by the EGFR on the same additional sites . Collectively, these data suggest that the interaction of Galpha(s) with residues 645-657 of the EGFR, or a peptide corresponding to this sequence alters the conformation of the G protein and/or the EGFR such that Galpha(s) is readily phosphorylated by the EGFR . The peptide EGFR-14, which does not activate Galpha(s), does not allow for the efficient phosphorylation of the G protein even though it does elevate the intrinsic tyrosine kinase activity of the EGFR . The hyperphosphorylation of Galpha(s) by EGFR is likely to require the contact of the G protein with EGFR-13 region (aa 645-657 in the EGFR) as well as augmentation of EGFR kinase activity. Arch Biochem Biophys, 2000 Nov 15, 383(2), 281 - 7 Optimization of expression of human sulfite oxidase and its molybdenum domain; Temple CA et al.; The conditions for the heterologous expression of both untagged and His-tagged human sulfite oxidase in Escherichia coli have been optimized . Maximum production of active enzyme requires expression in a mob- cell strain at low levels of the inducer . Using these conditions, 3.9-5.6 mg of untagged and 15 mg of His-tagged sulfite oxidase were isolated per liter of cell culture . These represent significantly higher levels than previously reported for any molybdopterin-containing protein . High levels of enzyme activity and molybdenum incorporation were maintained despite the increase in yield, and no significant differences in kinetic properties were observed between the tagged and untagged sulfite oxidase . Additionally, the molybdenum domain of sulfite oxidase was expressed in a stable, active form as a His-tagged protein . The molybdenum domain was also expressed in the presence of tungstate to enable examination of the molybdopterin-tungsten form of sulfite oxidase. Arch Biochem Biophys, 2000 Nov 15, 383(2), 272 - 80 Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani; Yang X et al.; The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli . Recombinant L . donovani AdoHcy hydrolase was then purified from cell-free extracts of E . coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange) . The purified recombinant L . donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit . Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L . donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium . The dissociation constant (Kd) for NAD+ with the L . donovani enzyme was estimated to be 2.1 +/- 0.2 microM . The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively . Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L . donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme . Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents. Arch Biochem Biophys, 2000 Nov 15, 383(2), 265 - 71 Properties of the nuclear proteasome activator PA28gamma (REGgamma); Wilk S et al.; PA28 or 11S REG is a proteasome activator composed of homologous alpha- and beta-subunits and predominantly found in the cytosol . A homologous protein originally known as the Ki antigen but now called PA28gamma or REGgamma is predominantly localized in the nucleus . To further characterize the biochemical properties of PA28gamma, we expressed and purified homogenous recombinant human protein with and without an N-terminal 6-His extension . PA28gamma is a heptamer based on the molecular masses of the native and monomeric proteins . The heptameric 6-His fusion protein can dimerize . Recombinant PA28y stimulates the proteasome-mediated hydrolysis of synthetic substrates containing hydrophobic, basic, and acidic amino acids in the P1 position . Stimulation is dependent on substrate size . PA28y only minimally stimulates degradation of the oxidized B chain of insulin . PA28gamma may facilitate the later stages of protein metabolism in the nucleus and/or have a more specialized role in controlling the levels of biologically active peptides in the nucleus. Arch Biochem Biophys, 2000 Nov 15, 383(2), 246 - 55 Reaction mechanism of electron transfer from FeII(CN)6(4-) or W(IV)(CN)8(4-) to the cupric ions in human copper, zinc superoxide dismutase; Hirose J et al.; The electron transfer reactions from FeII(CN)6(4-) and W(IV)(CN)8(4-) to the cupric ions in human copper, zinc superoxide dismutase were followed by the micro-stopped-flow method . The kinetic rate data clearly indicate that FeII(CN)6(4-) or W(IV)(CN)8(4-) first forms an adduct with the enzyme through the interaction with Arg143 of the active cavity and then an electron from FeII(CN)6(4-) or W(IV)(CN)8(4-) of the adduct transfers to the cupric ion in the enzyme . The dissociation constants of the adducts of FeII(CN)6(4-) and W(IV)(CN)8(4-) were 4.0(+/-0.3) x 10(-3) and 2.2(+/-0.3) x 10(-3) M, respectively . In spite of the difference between the standard redox potentials of FeIII(CN)6(3-)/FeII(CN)6(4-) (468 mV) and W(V)(CN)8(3-)/W(IV)(CN)8(4-) (556 mV), the electron transfer rate constant (0.148(+/-0.005) s(-1) of FeII(CN)6(4-) at 25 degrees C is very similar to that of W(IV)(CN)8(4-) (0.072(+/-0.011) s(-1)) . The entropy values of the adduct formations and the activation energies of the electron transfer rates were determined by the temperature dependence of the dissociation constants of the adducts and the electron transfer rates . The enthalpy values of the formation of adducts are almost zero, so that the driving forces to form the adducts are mainly derived from the entropy . The activation energy of the electron transfer rate of FeII(CN)6(4-) is very similar to that of W(IV)(CN)8(4-) . The formation of the adduct between FeII(CN)6(4-) and the enzyme was inhibited by the presence of various anions (ClO4-, SO4(2-), SCN-, and N3-) . The bulky anions SO4(2-) and ClO4- behave as competitive inhibitors for FeII(CN)6(4-); these anions should interact mainly with Arg143, as it has a positive charge at the entrance of the active cavity . The competitive inhibition constants of ClO4-, SO4(2-), and SCN- were 0.010, 0.012, and 0.008 M . The azide ion, which is smaller than SO4(2-) or ClO4-, shows mixed inhibition, because N3- can interact with Arg143 (competitive inhibition) and also directly binds to the cupric ion in h-SOD (noncompetitive inhibition) . The competitive and noncompetitive inhibition constants of N3- were 0.004 and 0.016 M, respectively. Arch Biochem Biophys, 2000 Nov 15, 383(2), 233 - 7 Two expressed soybean genes with high sequence identity to tomato Pti1 kinase lack autophosphorylation activity; Staswick P; An important signaling pathway for disease resistance in tomato involves the R gene product Pto which phosphorylates Ptil, a downstream member of this signaling cascade . Both Pto and Pti1 are Ser/Thr protein kinases capable of autophosphorylation in vitro . Two soybean (Glycine max L . Merr . var . Hobbit) cDNAs (sPti1a and sPti1b) were cloned and sequenced and found to each have 78% amino acid sequence identity with tomato Pti1 . Glutathione S-transferase fusions of sPti1a and b expressed in Escherichia coli did not autophosphorylate in vitro, but were efficiently phosphorylated by tomato Pto . Replacement of Tyr197 with an Asp that is invariant at this position in other protein kinases did not restore autophosphorylation activity to sPti1a or b . Tyr197 was also present in the Pti1 homologues of three distant relatives of G . max . Together these results suggest that soybean Pti1 might function in a Pto-like signaling pathway that does not require Pti1 kinase activity. Arch Biochem Biophys, 2000 Nov 15, 383(2), 215 - 24 Thermal cycling aids folding of a recombinant human beta-casein with four extra N-terminal amino acid residues; Hu Y et al.; Due to the limited secondary structure, it is believed that the caseins of milk, particularly the beta-caseins (beta-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues . However, the self-association of the human proteins with increasing temperature (T) and in the presence of Ca2+ is reproducible, implying that they normally fold into fixed tertiary structures . A nonphosphorylated recombinant human beta-CN with four extra amino acids at the N-terminus (GSHM-) was prepared and studied by laser light scattering, analytical ultracentrifugation, fluorescence spectroscopy, turbidity, and circular dichroism . In 3.3 M urea or at 4 degrees C, the protein was monomeric, as expected . Increasing T both without and with the addition of Ca2+ ions caused self-association as it does for the nonphosphorylated native beta-CN but with a somewhat different interaction pattern . However, returning the protein to its monomeric state by reequilibration at 4 degrees C followed again by increasing T caused a shift in the pattern . Such thermal cycling eventually caused the protein to equilibrate to a particular conformation where no more change could be observed . The resulting interaction pattern was similar to that of the native protein but differed particularly in that there was more extensive self-association for the recombinant mutant . The equilibration to a stable conformation was more rapid in the presence of Ca2+ ions . This suggests that the native protein normally folds into a particular conformation which may be aided by Ca2+ in the mammary gland . Further study of a recombinant form with the native amino acid sequence is needed. J Mol Biol, 2000 Oct 6, 302(5), 1139 - 51 Structural analysis of the transcriptional activation on Fis: crystal structures of six Fis mutants with different activation properties; Cheng YS et al.; The Fis protein regulates gene expression in Escherichia coli by activating or repressing transcription of a variety of genes . Fis can activate transcription when bound to DNA upstream of the RNA-polymerase-binding site, such as in the rrnB P1 promoter, or when bound to a site overlapping the -35 RNA polymerase binding site, such as in the proP P2 promoter . It has been suggested that transcriptional activation in both promoters results from interactions between specific amino acids within a turn connecting the B and C helices (the BC turn) in Fis and the C-terminal domain of the alpha-subunit of RNA polymerase (alphaCTD of RNAP) . Here, crystal structures of six Fis BC turn mutants with different transcriptional activation properties, Q68A, R71Y, R71L, G72A, G72D and Q74A, were determined at 1.9 to 2.8 A resolution . Two of these mutants, R71Y and R71L, crystallized in unit cells which are different from that of wild-type Fis, and the structure of R71L offers the most complete Fis model to date in that the extended structure of the N-terminal region is revealed . The BC turn in all of these mutant structures remains in a nearly identical gamma gamma beta-turn conformation as present in wild-type Fis . Analyses of the molecular surfaces of the transactivation region of the mutants suggest that several residues in or near the BC turn, including Gln68, Arg71, Gly72 and Gln74, form a ridge that could contact the alphaCTD of RNAP on one side . The structures and biochemical properties of the mutants suggest that Arg71 is the most critical residue for contacting RNAP within this ridge and that the glycine at position 72 helps to stabilize the structure. Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 435 - 40 A complex signaling module governs the activity of MalT, the prototype of an emerging transactivator family; Danot O; MalT, the specific activator of the maltose regulon, is the prototype of a family of high-molecular-mass ATP-binding bacterial transcription activators . On binding of its two positive effectors, the inducer maltotriose and ATP, MalT oligomerizes to an active state competent for promoter binding and transcription activation . In addition to its previously known DNA-binding domain, limited proteolysis showed that MalT contains three other domains, the boundaries of which were accurately delimited by N-terminal microsequencing . The N-terminal domain alone binds ATP . Maltotriose binding involves an extended region corresponding to domains 2 and 3, although weak binding to domain 3 alone was also observed . Moreover, maltotriose binding induces a conformational shift involving a movement of both domains 1 and 3 with respect to domain 2, leading to the active form of the protein . Sequence examination of the MalT homologues suggests that these three domains might constitute a signaling module. Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 427 - 31 The role of rigidity in DNA looping-unlooping by AraC; Harmer T et al.; We applied two experiments useful in the study of ligand-regulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli l-arabinose operon . In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites . The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences . Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains . The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose . When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains . These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected. J Bacteriol, 2001 Feb, 183(3), 1106 - 9 Preferential cleavage of degradative intermediates of rpsT mRNA by the Escherichia coli RNA degradosome; Spickler C et al.; RNase E, the principal RNase capable of initiating mRNA decay, preferentially attacks 5'-monophosphorylated over 5'-triphosphorylated substrates . Site-specific cleavage in vitro of the rpsT mRNA by RNase H directed by chimeric 2'-O-methyl oligonucleotides was employed to create truncated RNAs which are identical to authentic degradative intermediates . The rates of cleavage of two such intermediates by RNase E in the RNA degradosome are significantly faster (2.5- to 8-fold) than that of intact RNA . This verifies the preference of RNase E for degradative intermediates and can explain the frequent "all-or-none" behavior of mRNAs during the decay process. J Bacteriol, 2001 Feb, 183(3), 1085 - 9 umuDC-dnaQ Interaction and its implications for cell cycle regulation and SOS mutagenesis in Escherichia coli; Sutton MD et al.; The Escherichia coli SOS-regulated umuDC gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis . Specific interactions involving the UmuD and UmuD' proteins, both encoded by the umuD gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the umuDC gene products . Here we show that overproduction of the epsilon proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the umuDC gene products . Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD' and the C-terminal domain of epsilon. J Bacteriol, 2001 Feb, 183(3), 1038 - 46 Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C(1) and C(2) compounds in the methylotroph Methylobacterium extorquens AM1; Korotkova N et al.; Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1 . Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC) . phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3 . phaC was unlinked to phaA and phaB . Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB) . These genes show a high level of identity with each other at both DNA and amino acid levels . In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified . Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling . Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate . However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis . Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation . These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp. J Bacteriol, 2001 Feb, 183(3), 1022 - 31 Cooperativity between KorB and TrbA repressors of broad-host-range plasmid RK2; Zatyka M et al.; The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer . trbBp is the primary promoter responsible for expression of mating pair formation genes . We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the -10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp . Deletion analysis of TrbA showed that the C-terminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB . Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators. J Bacteriol, 2001 Feb, 183(3), 989 - 96 Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase; Klem TJ et al.; A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases . The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose . The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit . The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site . This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase . The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation . For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate . This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process . The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites. J Bacteriol, 2001 Feb, 183(3), 980 - 8 Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli; Bessette PH et al.; We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm . DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome . The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA . No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed . These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential . However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds . The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined . A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity . A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained . These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse . Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space. J Bacteriol, 2001 Feb, 183(3), 915 - 20 Diel expression of cell cycle-related genes in synchronized cultures of Prochlorococcus sp . strain PCC 9511; Holtzendorff J et al.; The cell cycle of the chlorophyll b-possessing marine cyanobacterium Prochlorococcus is highly synchronized under natural conditions . To understand the underlying molecular mechanisms we cloned and sequenced dnaA and ftsZ, two key cell cycle-associated genes, and studied their expression . An axenic culture of Prochlorococcus sp . strain PCC 9511 was grown in a turbidostat with a 12 h-12 h light-dark cycle for 2 weeks . During the light periods, a dynamic light regimen was used in order to simulate the natural conditions found in the upper layers of the world's oceans . This treatment resulted in strong cell cycle synchronization that was monitored by flow cytometry . The steady-state mRNA levels of dnaA and ftsZ were monitored at 4-h intervals during four consecutive division cycles . Both genes exhibited clear diel expression patterns with mRNA maxima during the replication (S) phase . Western blot experiments indicated that the peak of FtsZ concentration occurred at night, i.e., at the time of cell division . Thus, the transcript accumulation of genes involved in replication and division is coordinated in Prochlorococcus sp . strain PCC 9511 and might be crucial for determining the timing of DNA replication and cell division. J Bacteriol, 2001 Feb, 183(3), 909 - 14 Transfer protein TraY of plasmid R1 stimulates TraI-catalyzed oriT cleavage in vivo; Karl W et al.; The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated . As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins . The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY . Thus, the TraY and TraM proteins