Acta Chir Hung, 1997, 36(1-4), 152 - 3 Effects of antiendothelin treatment on the early hemodynamic changes in hyperdynamic endotoxemia; Kaszaki J et al.; We have performed a series of experiments to study the effects of a newly developed antisense homology box-derived endothelin (ET) antagonist peptide (ETR-P1/fl) on the early hemodynamic changes in a hyperdynamic endotoxemic dog model . Mean arterial pressure (MAP), cardiac output (CO) and myocardial contractility (MC) were measured in closed-chest animals . Plasma levels of ET-1,2 were determined by radioimmunassay . A hyperdynamic circulatory response was elicited with a 2-hour infusion of 5.3 micrograms/kg of E . coli endotoxin (ETX) . Control and ETX-treated animals received an infusion of ETR-P1/fl (0.1 mg/kg) i.v . ETX treatment decreased MAP and MC, increased initially CO, and a long lasting elevation in the plasma ET level was observed . In ETX-treated animals the administration of ETR-P1/fl significantly prolonged the increase in CO and inhibited the depression of MC . Our results suggest that treatment with the ET antagonist ETR-P1/fl may be advantageous in the early phase of endotoxemia. Circulation, 1997 Nov 4, 96(9 Suppl), II - 173-8 Recombinant endothelial nitric oxide synthase-transduced human saphenous veins: gene therapy to augment nitric oxide production in bypass conduits; Cable DG et al.; BACKGROUND: Nitric oxide is a potent vasodilator that also inhibits platelet aggregation and smooth muscle cell proliferation, properties that may prevent early and late occlusion of saphenous vein coronary bypass conduits . We determined whether human saphenous veins can be transduced with adenovirus vector-encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS), resulting in functional expression of recombinant nitric oxide synthase . METHODS AND RESULTS: Harvested segments of human saphenous vein were exposed for 1 hour at 37 degrees C to replication-deficient Ad.CMVeNOS (5 x 10(9) PFU/mL) or control adenovirus-encoding Escherichia coli beta-galactosidase (Ad.CMVLacZ; 5 x 10(9) PFU/mL) . The vein segments were analyzed for recombinant endothelial nitric oxide synthase expression and activity 48 hours later . Histochemical staining for recombinant beta-galactosidase activity was localized to the luminal endothelium and adventitia of vein segments transduced with Ad.CMVLacZ . Similarly, immunohistochemical staining with a monoclonal antibody for nitric oxide synthase localized recombinant gene expression to endothelial and adventitial cells in Ad.CMVeNOS veins; only endogenous nitric oxide synthase was identified in the endothelium of Ad.CMVLacZ veins . Nitrite generation after stimulation with calcium ionophore increased in Ad.CMVeNOS veins (1420.0+/-298.2 nM/mg versus 130.3+/-19.9 nM/mg; n=3; P<.05) . Isometric tension recording demonstrated augmented maximal relaxation to calcium ionophore (32+/-4.5% versus 17.4+/-7.4%; n=6; P<.05) after precontraction with norepinephrine . Bioassay superfusion demonstrated a twofold augmentation of the biodetector ring relaxation during calcium ionophore stimulation of Ad.CMVeNOS veins . CONCLUSIONS: Adenovirus-mediated gene transfer to human saphenous veins resulted in functional transgene expression with increased nitric oxide release . These or similar molecular techniques to increase nitric oxide production may reduce the risk of early thrombosis in saphenous vein grafts. South Med J, 1997 Nov, 90(11), 1065 - 8 Necrotizing fasciitis: improved survival with early recognition by tissue biopsy and aggressive surgical treatment; Majeski J et al.; BACKGROUND: Necrotizing fasciitis is a soft tissue gangrenous infection that is optimally treated by early diagnosis, radical surgical debridement of all involved necrotic tissue, broad spectrum antibiotics, and aggressive nutritional support . The early clinical diagnosis of an area of necrotizing fasciitis is difficult and frequently unreliable . We are reporting a series of cases in which an early, accurate diagnosis of necrotizing fasciitis was established by a frozen section tissue biopsy obtained at the bedside . METHODS: Over a 15-year period, a consecutive series of 43 patients had a bedside biopsy under local anesthesia with immediate frozen section evaluation . All patients were seen in the hospital or emergency room for treatment of an inflammatory process . RESULTS: These 43 patients had bedside biopsy and frozen section evaluation of an inflammatory process . Twelve patients were found to have necrotizing fasciitis . These patients were treated with immediate surgical debridement of all gross necrotic tissue, broad spectrum antibiotics, and adequate nutritional support . All of them survived . No cases of infectious gangrene occurred in the group of patients whose biopsy did not reveal necrotizing fasciitis . CONCLUSION: Frozen section tissue biopsy is a useful adjunct in establishing an early, accurate diagnosis of infectious gangrene. Brain Res Brain Res Protoc, 1997 Aug, 1(3), 307 - 19 Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits; Beattie RE et al.; Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue . The comparative distribution of these in normal human brain is largely unknown . The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain . Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E . coli . Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns . The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits. J Capillary Electrophor, 1996 Nov-Dec, 3(6), 301 - 7 Purity testing of recombinant proteins by capillary electrophoresis; Kundu S et al.; Purity testing of recombinant DNA (rDNA) proteins using slab gel electrophoresis in conjunction with scanning densitometry is time consuming and labor intensive and is difficult to reproduce because the dyes used for visualizing proteins do not bind in a stoichiometric fashion for all proteins . The present report describes a micellar capillary zone electrophoresis (MCZE) procedure that overcomes these difficulties . The MCZE method was evaluated to estimate protein purity of hydrophobic cytomegalovirus proteins, expressed E . coli, and highly glycosylated hepatitis C virus proteins, expressed in Chinese hamster ovary cells . The results obtained by the MCZE procedure correlated very well with the purity results quantitated by the conventional slab gel electrophoresis method using purified Coomassie Brilliant Blue dye to reduce anomalies . MCZE may serve as an alternative method for in-process and purity testing of rDNA proteins. J Capillary Electrophor, 1997 Jan-Feb, 4(1), 7 - 13 Capillary electrophoresis for purity estimation and in-process testing of recombinant GB virus-C proteins; Kundu S et al.; Protein purity estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with scanning densitometry is a critical component in the manufacture of recombinant proteins for commercial diagnostic assays . However, the procedure is time consuming and often difficult to reproduce because commercial dyes that are used for visualizing proteins do not bind in a stoichiometric fashion for all proteins . The present report describes the use of a rapid and dye-independent SDS polymer-filled capillary gel electrophoresis (CE-SDS) method to estimate protein purity . The CE-SDS method was used for in-process and final purity testing of GB virus-C (GBV-C) fusion proteins produced in E . coli, and was directly compared with the conventional SDS-PAGE method using purified Coomassie blue dye to reduce protein staining anomalies . The CE-SDS method may serve as an alternative or replacement method to the lengthy and tedious SDS-PAGE method . This study also demonstrates that the observed molecular weight of the fusion protein, determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), provides higher accuracy than values estimated by either CE-SDS or SDS-PAGE methods. Biochim Biophys Acta, 1997 Oct 24, 1361(3), 229 - 34 Iron potentiates nitric oxide scavenging by dithiocarbamates in tissue of septic shock mice; Komarov AM et al.; Vanin and co-workers (Kubrina et al., Biochim . Biophys . Acta 1176 (1993) 240-244; Mikoyan et al., Biochim . Biophys . Acta 1269 (1995) 19-24) reported that short term (30 min) iron (Fe) exposure potentiates nitric oxide (NO) production in tissues of septic shock mice, based on increased formation of NO complex by diethyldithiocarbamate (DETC) . We have reexamined the effect of Fe administration in mice treated with Escherichia coli lipopolysaccharide (LPS) and have not found any changes in nitrosylhemoglobin (HbNO) or (NOs- + NO3-) levels in blood 30 min after Fe-citrate complex injection . However, Fe-citrate promotes NO complex formation by iron-dependent NO traps: DETC, pyrrolidinedithiocarbamate (PDTC) and N-methyl-D-glucamine dithiocarbamate (MGD), when given simultaneously at 6 h after LPS . Rather than potentiation of NO production, our data support that short-term iron treatment (30 min) enhances in vivo spin trapping ability of dithiocarbamate. J Thorac Cardiovasc Surg, 1997 Nov, 114(5), 793 - 801; discussion 801-2 Successful in vivo and ex vivo transfection of pulmonary artery segments in lung isografts; Yano M et al.; OBJECTIVE: Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection . Efficient gene transfection to the whole organ may prove problematic . Proximal pulmonary artery endothelial transfection might provide beneficial downstream effects on the whole graft . The aim of this study was to determine the feasibility of transfecting proximal pulmonary artery segments in lung isografts . METHODS: Male Fischer rats were divided into six groups . In vivo transfection: In group I (n = 7), a proximal segment of the left pulmonary artery was isolated and injected with saline solution by means of a catheter inserted through the right ventricle . After an exposure period of 20 minutes, clamps were removed and blood flow was restored . In group II (n = 7), the isolated arterial segments were injected with adenovirus carrying the Escherichia coli LacZ gene encoding for beta-galactosidase . Ex vivo transfection: In group III (n = 5), arterial segments were injected ex vivo with saline solution and in group IV (n = 5) with the adenovirus construct . In group V (n = 6), arteries were injected with saline solution and in group VI (n = 11) with liposome chloramphenicol acetyl transferase cDNA . In groups I to IV, animals were killed on postoperative day 3 and transgene expression was assessed by Bluo-Gal staining . In groups V and VI, animals were killed on postoperative day 2 and transgene expression was assessed by chloramphenicol acetyl transferase activity assay . RESULTS: Transgene expression was detected grossly and microscopically in endothelial and smooth muscle cells of pulmonary artery segments from all surviving animals of groups II and IV . In group VI, chloramphenicol acetyl transferase activity was significant in all assessed arterial segments . CONCLUSION: Significant transgene expression is observed in proximal pulmonary artery segments after both in vivo and ex vivo exposure. Am J Emerg Med, 1997 Nov, 15(7), 644 - 7 Arterial oxygen desaturation during emergent nonsedated upper gastrointestinal endoscopy in the emergency department; Yen D et al.; A prospective study was conducted to see whether emergent esophagogastroduodenoscopy (EGD) in patients with active upper gastrointestinal (GI) bleeding is associated with more oxygen desaturation than nonemergent EGD . Emergent EGD was performed in the study patients with active upper GI bleeding . Nonemergent EGD was performed in the control patients . Determination of oxygen saturation (Sao2) was measured by pulse oximeter . A decrease in Sao2 of > 4% was more frequent in the study patients (26%, 13 of 50) than in controls (6%, 3 of 50) (P < .01) . During EGD, mean oxygen saturation decreased significantly in both groups of patients . After EGD, mean oxygen saturation did not recover toward the pre-endoscopy insertion level in the study group (P < .01) . A linear association was found that oxygen desaturation = 5.46 + 0.15 (status) -0.06 (baseline oxygen saturation) . Emergent EGD for active upper GI bleeding in the emergency department tends to be associated with more frequent significant oxygen desaturation than nonemergent EGD . Continuous oxygen supplementation and oxygen saturation monitoring may be used during emergent nonsedated EGD in the emergency department. Phytochemistry, 1997 Nov, 46(5), 801 - 4 Synthesis of cytokinin glucuronides for the selection of transgenic plant cells; Okkels FT et al.; Glucuronide derivatives of cytokinins have been synthesized for use as agents for the selection of plant cells transformed with a beta-glucuronidase (GUS) gene . In this selection system, the GUS gene functions as both a selectable, as well as a screenable gene . GUS liberates active cytokinin from inactive cytokinin glucuronides which then stimulates growth and regeneration of the transformed cells . The frequently used cytokinin N6-benzyladenine was conjugated to glucuronic acid at N-3 or at N-9 but only the former was a substrate for GUS . The glucuronide of isopentenyladenine was also made, by coupling at the N-3 . This compound was readily hydrolysed by GUS. Virology, 1997 Nov 10, 238(1), 79 - 84 Crystals of Rous sarcoma virus capsid protein show a helical arrangement of protein subunits; Kovari LC et al.; Crystals of Rous sarcoma virus (RSV) capsid protein diffract X rays to 3.5 A resolution and belong to the monoclinic space group C2 with unit cell parameters a = 374.4 A, b = 128.1 A, c = 200.2 A, and beta = 121.8 degrees . One asymmetric unit of the crystal may contain between 28 and 35 molecules, based on reasonable crystal density assumptions . A self-rotation function and Patterson synthesis suggest that RSV capsid protein crystallizes as a helical array . The determinants of the viral particle morphology are not encoded in the capsid alone . The assembly of a helical array in the crystal reflects the absence of any conformational switching . However, it is expected that the subunit interactions seen in the crystal will be preferred and will relate to those found in the immature or mature virion. Am J Physiol, 1997 Nov, 273(5 Pt 1), L967 - 79 cAMP and purinergic P2y receptors upregulate and enhance inducible NO synthase mRNA and protein in vivo; Greenberg SS et al.; Adenosine 3',5'-cyclic monophosphate (cAMP) and purinergic P2y receptor agonists upregulate inducible nitric oxide (NO) synthase (iNOS) but inhibit Escherichia coli endotoxin lipopolysaccharide (LPS)- and cytokine-mediated upregulation of iNOS in cultured cells . We examined the effects of cAMP and P2y receptor agonists on the iNOS system in vivo . Intratracheal administration of dibutyryl-cAMP (DBcAMP, 0.1 and 1 mg/kg), a P2y receptor agonist {2-methylthioadenosine 5'-triphosphate (MeS-ATP), 5 mg/kg}, or LPS (0.6 mg/kg) to rats 2 h before bronchoalveolar lavage (BAL) increased iNOS mRNA (competitor-equalized reverse transcription-polymerase chain reaction) and iNOS protein (Western blot) in rat alveolar macrophages compared with the effects of sterile phosphate-buffered saline (0.5 ml it) . At equal levels of upregulation of iNOS mRNA, 1) LPS, but not DBcAMP or MeS-ATP, upregulated nuclear transcription factor-kappa B (NF-kappa B) and 2) iNOS protein and formation of NO were greater in alveolar macrophages from LPS- and MeS-ATP-treated rats than from DBcAMP-treated rats . Administration of DBcAMP or MeS-AMP 15 min before LPS did not inhibit LPS-induced alveolar macrophage-derived iNOS mRNA, iNOS protein, and NO . Diethyldithiocarbamate (DETC, 5 mg/kg it) inhibited LPS-induced iNOS mRNA but did not affect upregulation of iNOS mRNA produced by the other agonists . We conclude that an LPS-dependent and -independent pathway of iNOS mRNA induction exists in vivo . The former is activated by IPS and most cytokines, is associated with upregulation of NF-kappa B and inhibited by DETC, and elicits an inflammatory response . The latter, activated by DBcAMP and MeS-ATP, is not associated with upregulation of NF-kappa B, inhibition by DETC, or activation of inflammation . The two systems are additive in vivo rather than antagonistic . Speculatively, if the LPS-independent iNOS pathway exists in humans, the iNOS in tissues from patients taking drugs affecting cAMP or P2y receptors may be iatrogenic rather than pathogenetic in origin. Am J Physiol, 1997 Nov, 273(5 Pt 1), L930 - 40 Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle; Larsen JK et al.; The 27-kDa heat shock protein (HSP27) is expressed in a variety of tissues in the absence of stress and is thought to regulate actin filament dynamics, possibly by a phosphorylation/dephosphorylation mechanism . HSP27 has also been suggested to be involved in contraction of intestinal smooth muscle . We have investigated phosphorylation of HSP27 in airway smooth muscle in response to the muscarinic agonist carbachol . Carbachol increased 32P incorporation into canine tracheal HSP27 and induced a shift in the distribution of charge isoforms on two-dimensional gels to more acidic, phosphorylated forms . The canine HSP27 amino acid sequence includes three serine residues corresponding to sites in human HSP27 known to be phosphorylated by mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2 . To determine whether muscarinic receptors are coupled to a "stress response" pathway in smooth muscle culminating in phosphorylation of HSP27, we assayed MAPKAP kinase-2 activity and tyrosine phosphorylation of p38 mitogen-activated protein (MAP) kinase, the enzyme thought to activate MAPKAP kinase-2 . Recombinant canine HSP27 expressed in Escherichia coli was a substrate for MAPKAP kinase-2 in vitro as well as a substrate for endogenous smooth muscle HSP27 kinase, which was activated by carbachol . Carbachol also increased tyrosine phosphorylation of p38 MAP kinase . SB-203580, an inhibitor of p38 MAP kinases, reduced activation of endogenous HSP27 kinase activity and blocked the shift in HSP27 charge isoforms to acidic forms . We suggest that HSP27 in airway smooth muscle, in addition to being a stress response protein, is phosphorylated by a receptor-initiated signaling cascade involving muscarinic receptors, tyrosine phosphorylation of p38 MAP kinase, and activation of MAPKAP kinase-2. Am J Physiol, 1997 Nov, 273(5 Pt 1), G1118 - 26 pH-dependent changes of nitric oxide, peroxynitrite, and reactive oxygen species in hepatocellular damage; Shu Z et al.; Low arterial blood pH and sustained nitric oxide (NO) production are critical parameters in inflammatory events such as sepsis, and appropriate treatment is still under debate . Because the stability of nitrogen and oxygen intermediates is dependent on the surrounding pH, we investigated whether the relationship among NO, peroxynitrite (ONOO-), and reactive oxygen species production also depends on the pH value, particularly with respect to their effects on hepatocellular damage . Our studies demonstrate that the extracellular pH influences NO and hydroxyl radical (OH) production in hepatocytes . Acidification (pH 7.0) of the medium revealed a significant increase (P < 0.05) of OH-like radicals, enhanced hepatocellular damage, and a sharp drop in cellular glutathione (GSH) content compared with levels measured at physiological or alkaline pH conditions . Furthermore, inhibition of NO synthesis at all pH conditions resulted in decreased NO production and cellular GSH levels but a simultaneous increase of OH-like radicals and hepatocellular damage with a maximum seen at pH 7.0 . Our results suggest that hepatocellular damage is in part regulated by the surrounding pH and that inhibition of NO synthesis at acidic conditions (e.g., in sepsis) leads to increased reactive oxygen-mediated cell injury. J Biol Chem, 1997 Nov 28, 272(48), 30463 - 9 RTX toxins recognize a beta2 integrin on the surface of human target cells; Lally ET et al.; Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coli alpha-hemolysin are RTX toxins that kill human immune cells . We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins . Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa . Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the beta2 integrin, lymphocyte function-associated antigen 1 (LFA-1) . Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis . Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells . Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins . These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins. J Biol Chem, 1997 Nov 28, 272(48), 30345 - 9 Conserved properties between functionally distinct MutS homologs in yeast; Pochart P et al.; In the yeast Saccharomyces cerevisiae there are five nuclear MutS homologs that act in two distinct processes . MSH2, 3, and 6 function in mismatch repair in both vegetative and meiotic cells, whereas MSH4 and MSH5 act specifically to facilitate crossovers between homologs during meiosis . Coimmunoprecipitation as well as two-hybrid experiments indicate that the Msh4 and Msh5 proteins form a hetero-oligomeric structure similar to what is observed for the Msh proteins involved in mismatch repair . Mutation of conserved amino acids in the NTP binding and putative helix-turn-helix domains of Msh5p abolish function but are still capable of interaction with Msh4p, suggesting that NTP binding plays a role downstream of hetero-oligomer formation . No hetero-oligomers are observed between the mismatch repair MutS proteins (Msh2p and Msh6p) and either Msh4p or Msh5p . These results indicate that one level of functional specificity between the mismatch repair and meiotic crossover MutS homologs in yeast is provided by the ability to form distinct hetero-oligomers. J Biol Chem, 1997 Nov 28, 272(48), 30228 - 36 The RepA protein of plasmid RSF1010 is a replicative DNA helicase; Scherzinger E et al.; The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication . RepA is one of the smallest known helicases . The protein forms a homohexamer of 29,896-Da subunits . A variety of methods were used to analyze the quaternary structure of RepA . Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction . These results agree with recent data obtained from RepA crystals diffracting at 3.5-A resolution (Roleke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W . (1997) Acta Crystallogr . Sec . D 53, 213-216) . The RepA helicase has 5' --> 3' polarity . As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3'-tail mimicking a replication fork . Optimal unwinding activity was achieved at the remarkably low pH of 5.5 . In the presence of Mg2+ (Mn2+) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP . UTP and dTTP are poor effectors . Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not . The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB' protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein. J Biol Chem, 1997 Nov 28, 272(48), 30047 - 53 Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase; Sawada K et al.; Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well . Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits . Expression of bcyt fused to glutathione S-transferase (GST) together with the delta subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed delta subunit from proteolytic attack in the cell . These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell . The affinity purified GST-bcyt did not contain significant amounts of delta, suggesting that the interaction of these subunits was relatively weak . Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits . The ATPase activity was reconstituted from the purified bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon . Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex . The result indicated that interaction of b and delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction. J Biol Chem, 1997 Nov 28, 272(48), 29998 - 30001 The role of the buried aspartate of Escherichia coli thioredoxin in the activation of the mixed disulfide intermediate; LeMaster DM et al.; The structurally homologous protein disulfide isomerases and thioredoxins exhibit a 10(5) variation of redox equilibria . It is demonstrated that the kinetic distinction among these protein family members lies primarily in the rate of breakdown of the mixed disulfide intermediate . The conserved buried acid group serves as a proton transfer catalyst for the buried active site cysteine in the formation and breakdown of the mixed disulfide . The reduction rate of Escherichia coli thioredoxin by dithiothreitol is directly proportional to the fraction of Asp-26 in the protonated form over the pH range of 6-9 . The kinetic role of Asp-26 is further probed via differential solvent kinetic isotope effect measurements versus a D26N variant . The differential solvent isotope effect of 0.6 is consistent with a direct proton donation to the thiolate leaving group (Cys-35) via an enforced general acid catalysis by trapping mechanism . Such a donation necessitates a structural rearrangement as these two buried side chains are separated by 6 A in both the oxidized and reduced forms of the protein. Eur J Surg, 1997 Oct, 163(10), 779 - 88 Growth hormone increases and IGF-1 reduces the response to Escherichia coli infusion in injured pigs; Unneberg K et al.; OBJECTIVE: To investigate if growth hormone (GH) or its main mediator insulin-like growth factor-1 (IGF-1) alters the response to infusion of live Escherichia coli in injured pigs . DESIGN: Controlled experiment . SETTING: University laboratory, Norway . SUBJECTS: 30 piglets . INTERVENTIONS: The response to infusion of Escherichia coli was compared after a bolus of GH 16 IU (n = 8) or a continuous infusion of IGF-1 1.3 mg/hour (n = 8) in injured piglets . A group with trauma (surgery) and Escherichia coli infusion (n = 8) and a group with trauma only (n = 6) served as controls . MAIN OUTCOME MEASURES: Systemic and regional haemodynamics, oxygen consumption, and acid-base regulation; and circulating concentrations of catecholamines, free fatty acids (FFA), glucose, and lactate Results: After infusion of Escherichia coli, cardiac output was lower and heart rate was higher in the GH than in the IGF-1-treated group . Aortic pH was lower in the GH group compared with the septic controls, whereas aortic pH was higher in the IGF-1 group compared with the septic controls . Portal vein pH was lower in the GH group than in the other three groups . Free fatty acids and lactate concentrations were higher in the GH group than in the other three groups . Glucose concentrations were lower in the IGF-1 group than in the other three groups . Renal artery flow was higher in the IGF-1 than in the GH group and the septic controls . Circulating concentrations of dopamine was higher in the IGF-1 group than in the other three groups, whereas that of noradrenaline was higher in the GH group than in the IGF-1 group . (For all differences stated, p < 0.05) . CONCLUSIONS: Acute treatment with GH increased the circulatory and metabolic response to Escherichia coli infusion, in contrast to treatment with IGF-1, which reduced the response FEBS Lett, 1997 Oct 27, 416(3), 349 - 52 FNR is a direct oxygen sensor having a biphasic response curve; Jordan PA et al.; FNR is a transcription regulator that controls the expression of target genes in response to anoxia . Anaerobiosis is accompanied by the acquisition of two {4Fe-4S}2+ clusters per FNR dimer and the ability to bind DNA site-specifically . Oxidation of the {4Fe-4S}2+ form of FNR by O2 produced a non-DNA-binding, transcriptionally inactive form which also contains an iron-sulfur cluster, recently identified by Mossbauer spectroscopy as a {2Fe-2S} cluster (Khoroshilova et al., 1997, PNAS . 94, 6078) . Complete conversion needed at least 2.5-3.0 molecules of O2 per {4Fe-4S}2+ cluster . Using sub-stoicheiometric amounts of air-saturated buffer, stable equilibria were established in which the {4Fe-4S}2+ and {2Fe-2S}2+ forms co-exist and no EPR detectable free ferric ions were released . In contrast, a 20-fold molar excess K3Fe(CN)6 was required to oxidise the {4Fe-4S}2+ cluster and in this case, ferric ions were released . FNR is therefore a sensitive O2 sensor. FEBS Lett, 1997 Oct 27, 416(3), 247 - 50 Glutamate-286 mutants of cytochrome bo-type ubiquinol oxidase from Escherichia coli: influence of mutations on the binuclear center structure revealed by FT-IR and EPR spectroscopies; Tsubaki M et al.; Glutamate-286 mutants of cytochrome bo-type ubiquinol oxidase from Escherichia coli were examined by EPR and FT-IR spectroscopies . We confirmed a very low enzymatic activity for E286Q . However, E286D retained one-third of the wild-type activity, probably due to the presence of the carboxylic group on the side-chain . The effect of the mutations at position 286 on the binuclear site was observed clearly in the EPR spectral change for the air-oxidized state . The effect was more significantly manifested in the presence of cyanide or azide in the oxidized state . In contrast, the mutations only slightly perturbed the binuclear center of the CO-reduced enzymes . These results indicate the importance of a direct through-bond connectivity between CuB and Glu286 via Pro285 and His284. Mol Cell Biol, 1997 Dec, 17(12), 7151 - 8 Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase; Xia SJ et al.; Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time . Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination . Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains . Expression of HsRAD51, a human homolog of the yeast RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells . Stable transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal recombination and the level of HsRAD51 transcripts by two- to fivefold . T-antigen induction of recombination was efficiently blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that HsRAD51 expression mediates augmented recombination . Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may block such inactivation and liberate HsRAD51 . Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality. Mol Cell Biol, 1997 Dec, 17(12), 6822 - 30 Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand; Bessho T et al.; Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template . Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged . To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link . We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands . The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link . This gap may act as a recombinogenic signal to initiate cross-link removal. Mol Cell Biol, 1997 Dec, 17(12), 6815 - 21 Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation; Deed RW et al.; The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins . These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences . Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E . Hara, M . Hall, and G . Peters, EMBO J . 16:332-342, 1997) . We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo . Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein . Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity . These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation. Mutat Res, 1997 Oct, 385(1), 41 - 6 Suppressing effects of S-methyl methanethiosulfonate and diphenyl disulfide on mitomycin C-induced somatic mutation and recombination in Drosophila melanogaster and micronuclei in mice; Nakamura YK et al.; S-Methyl methanethiosulfonate (MMTS) and diphenyl disulfide (DPDS) are temporary enzyme-sulfhydryl blocking agents . They are naturally occurring phytoalexin-like and synthetic substances known to be very potent bio-antimutagens in Escherichia coli B/r WP2 . In the present paper, the suppressing effects of MMTS on mitomycin C (MMC)-induced mutant wing spots in the somatic mutation and recombination test (SMART) of Drosophila melanogaster, and of MMTS and DPDS on MMC-induced micronucleated peripheral reticulocytes are described . MMTS consistently reduced the numbers of MMC-induced small single, large single and twin spots per wing at a dose of 10-1000 micrograms/vial, in a dose-dependent manner . MMTS reduced the number of twin spots per wing on the spontaneous mutation at the dose of 1000 micrograms/vial . MMTS and DPDS dose-dependently reduced the frequencies of MMC-induced micronucleated peripheral reticulocytes at a dose of 10-40, and 3-100 micrograms/kg, respectively . Our results confirmed that enzyme-sulfhydryl blocking agents, such as MMTS and DPDS, are effective antimutagens in vivo too. Biochemistry, 1997 Nov 18, 36(46), 14120 - 7 Ligand-induced movement of helix X in the lactose permease from Escherichia coli: a fluorescence quenching study; Wang Q et al.; Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues (Trp-less permease) . Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity . The intrinsic fluorescence emission of each single-Trp mutant does not change significantly after addition of beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside (TDG), indicating that ligand induces little change in the microenvironment of the Trp residues . However, fluorescence quenching studies with the brominated detergent 7,8-dibromododecyl beta,d-maltoside (BrDM) demonstrate that a Trp residue in place of Val315, Val326, or Val331 becomes less accessible to BrDM in the presence of TDG, while a Trp residue in place of Leu318 or Leu329 becomes more accessible . Acrylamide quenching studies with Leu318-->Trp and Val331-->Trp permeases or 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS)-labeled Thr320-->Cys and Glu325-->Cys permeases indicate that positions 318 and 325 also become more accessible to a hydrophobic environment in the presence of TDG, while positions 320 and 331 become less accessible . The findings are consistent with a recently proposed mechanism for energy coupling in lactose permease {Kaback, H . R . (1997) Proc . Natl . Acad . Sci . U.S.A . 94, 5539-5543} in which substrate binding causes a conformational change resulting in movement of Glu325 to a nonpolar environment with a dramatic increase in pKa. Biochemistry, 1997 Nov 18, 36(46), 14056 - 63 RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates; Kerr SG et al.; The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions . In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer . The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide . The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively . The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively . These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position . Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine . Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity . This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates . Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation. J Biol Chem, 1997 Nov 21, 272(47), 29919 - 26 Functional properties of the separate subunits of human DNA helicase II/Ku autoantigen; Ochem AE et al.; The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II) . HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions . To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli . The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A . (1994) EMBO J . 13, 4991-5001) . Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195) . The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits . The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends . The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity. J Biol Chem, 1997 Nov 21, 272(47), 29852 - 8 The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors; Inamoto S et al.; Octamer binding transcription factors (Oct factors) play important roles in activation of transcription of various genes but, in some cases, require cofactors that interact with the DNA binding (POU) domain . In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein . MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH) . Further analyses showed that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner . MAT1-containing TFIIH was also shown to interact with POU domains of Oct-1 and Oct-2 . MAT1 is shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2 . Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK . However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific, suggesting that, in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo . These results suggest (i) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (ii) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (iii) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions. J Biol Chem, 1997 Nov 21, 272(47), 29566 - 71 Alanine insertion scanning mutagenesis of lactose permease transmembrane helices; Braun P et al.; A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function . We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanning Escherichia coli inner membrane protein . Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport . A more detailed Ala insertion scan was made of transmembrane helix III . The results pin-point a central region of approximately 2 helical turns that is crucial for lactose permease stability and/or activity . A Trp scan in this region identified 2 residues essential for lactose permease stability . From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments. J Biol Chem, 1997 Nov 21, 272(47), 29442 - 8 The Ov20 protein of the parasitic nematode Onchocerca volvulus . A structurally novel class of small helix-rich retinol-binding proteins; Kennedy MW et al.; Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite . DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli . Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-alpha-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol . The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized beta-sheet-rich mammalian retinol- and fatty acid-binding proteins . Secondary structure prediction algorithms indicate that a alpha-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of beta structures, and this was confirmed by circular dichroism . The protein is highly stable in solution, requiring temperatures in excess of 90 degrees C or high denaturant concentrations for unfolding . Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes . The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy. Genes Dev, 1997 Nov 15, 11(22), 3072 - 82 Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed; Jimenez G et al.; Relatively little is known about the molecular mechanisms involved in transcriptional repression, despite its importance in development and differentiation . Recent evidence suggests that some transcriptional repressors act by way of adaptor molecules known as corepressors . Here, we use in vivo functional assays to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in the Drosophila embryo . Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix-loop-helix proteins . Our results indicate not only that repression by Hairy requires Gro, but that a repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent . The latter result correlates with an ability of this En domain to bind to Gro in vitro . In contrast, repressor regions from the Even-skipped, Snail, Kruppel, and Knirps transcription factors are effective in the absence of Gro . These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En. Fungal Genet Biol, 1997 Oct, 22(2), 92 - 102 Molecular characterization of mutants of the acetate regulatory gene facB of Aspergillus nidulans; Todd RB et al.; The facB gene of Aspergillus nidulans encodes a DNA binding transcriptional activator required for growth on acetate as a sole carbon source . FacB contains N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA binding and leucine zipper-like heptad repeat motifs and central and C-terminal acidic alpha-helical regions . facB recessive loss of function mutants are deficient in acetate induction of acetyl-CoA synthase, isocitrate lyase, malate synthase, acetamidase, and NADP-isocitrate dehydrogenase . Characterization of lesions in facB mutant alleles has localized important functional regions of the FacB protein . Two extreme mutants are shown to lack the C-terminal region of the protein . Two temperature sensitive mutants contain amino acid substitutions in the DNA binding domain and are shown to affect acetate induction of amdS-lacZ expression and confer temperature sensitive in vitro DNA binding . Two temperature sensitive facB mutations result in thermolability of acetyl-CoA synthase, isocitrate lyase, and malate synthase but not acetamidase or NADP-isocitrate dehydrogenase in crude extracts . This suggests that FacB may have a structural role in acetate metabolism in addition to its regulatory function . Arch Biochem Biophys, 1997 Nov 15, 347(2), 221 - 8 Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQO2), a dihydronicotinamide riboside dependent oxidoreductase; Wu K et al.; Human NAD(P)H:quinone acceptor oxidoreductase-2 (NQO2) has been prepared using an Escherichia coli expression method . NQO2 is thought to be an isoform of DT-diaphorase (EC 1.6.99.2) {also referred to as NAD(P)H:quinone acceptor oxidoreductase} because there is a 49% identity between their amino acid sequences . The present investigation has revealed that like DT-diaphorase, NQO2 is a dimer enzyme with one FAD prosthetic group per subunit . Interestingly, NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor . It catalyzes a two-electron reduction of quinones and oxidation-reduction dyes . One-electron acceptors, such as potassium ferricyanide, cannot be reduced by NQO2 . This enzyme also catalyzes a four-electron reduction, using methyl red as the electron acceptor . The NRH-methyl red reductase activity of NQO2 is 11 times the NADH-methyl red reductase activity of DT-diaphorase . In addition, through a four-electron reduction reaction, NQO2 can catalyze nitroreduction of cytotoxic compound CB 1954 {5-(aziridin-1-yl)-2,4-dinitrobenzamide} . NQO2 is 3000 times more effective than DT-diaphorase in the reduction of CB 1954 . Therefore, NQO2 is a NRH-dependent oxidoreductase which catalyzes two- and four-electron reduction reactions . NQO2 is resistant to typical inhibitors of DT-diaphorase, such as dicumarol, Cibacron blue, and phenindone . Flavones are inhibitors of NQO2 . However, structural requirements of flavones for the inhibition of NQO2 are different from those for DT-diaphorase . The most potent flavone inhibitor tested so far is quercetin (3,5,7,3',4'- . 6pentahydroxyflavone) . It has been found that quercetin is a competitive inhibitor with respect to NRH (Ki = 21 nM) . NQO2 is 43 amino acids shorter than DT-diaphorase, and it has been suggested that the carboxyl terminus of DT-diaphorase plays a role in substrate binding (S . Chen et al., Protein Sci . 3, 51-57, 1994) . In order to understand better the basis of catalytic differences between NQO2 and DT-diaphorase, a human NQO2 with 43 amino acids from the carboxyl terminus of human DT-diaphorase (i.e., hNQO2-hDT43) has been prepared . hNQO2-hDT43 still uses NRH as an electron donor . In addition, the chimeric enzyme is inhibited by quercetin but not dicumarol . These results suggest that additional region(s) in these enzymes is involved in differentiating NRH from NAD(P)H . Arch Biochem Biophys, 1997 Nov 15, 347(2), 181 - 92 Biological oxidations and P450 reactions . Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1; Savas U et al.; Orthologs of a previously identified CYP1B subfamily designated CYP1B1, which are constitutively expressed in mammary, uterine, and embryonic cells, have previously been functionally linked to 7,12-dimethylbenz-a-anthracene (DMBA) metabolism . A chimeric construct of mouse CYP1B1 in which the 20 NH2-terminal amino acids have been replaced by eight residues from human CYP17 has been expressed in Escherichia coli . This recombinant mouse CYP1B1 (recCYP1B1m) exhibited DMBA metabolism accurately reproducing the characteristic product distribution and specific activity of 3.4 nmol/nmol P450/min seen in C3H10T1/2 cells from which this cDNA has been cloned . The high proportion of 10,11- and 3,4-dihydrodiols and near absence of 5,6-dihyrodiol- and 7-hydroxy-DMBA metabolites are seen only in rodent microsomes where CYP1B1 is highly expressed . This distribution of products from recCYP1B1m was highly dependent on addition of epoxide hydrolase, particularly the ratio of 3,4-dihydrodiol to 4-phenol metabolites . These characteristics in addition to inhibition by antibodies raised to recCYP1B1m establish that the CYP1B1 cDNA indeed encodes the P450 responsible for polycyclic aromatic hydrocarbon (PAH) metabolism from C3H10T1/2 cells . DMBA metabolites from cDNA-expressed human CYP1B1 (recCYP1B1h) however, exhibited a different regioselectivity toward DMBA resembling human CYP1A1 catalyzed DMBA metabolism . Reconstitution of recCYP1B1m with different concentrations of NADPH-P450 reductase indicated a high affinity interaction with an apparent Km of 3 nM . Large PAH such as benz{a}pyrene, benz{e}pyrene, benz{a}anthracene, DMBA, 3-methylcholanthrene, and 1-ethynylpyrene bound to recCYP1B1m with high affinity (Kd 0.08 to 0.22 microM) concomitant with substantial spectral shifts (40% low to high spin state change) . Smaller PAHs like pyrene, phenanthrene, and naphthalene neither produced spectral changes nor inhibited the spectral change caused by benz{a}pyrene . Among tested steroids, progesterone bound weakly to recCYP1B1m (Kd > 20 microM) with a comparable spectral shift and was a weak inhibitor of DMBA metabolism, but was not metabolized . While 17beta-estradiol is a substrate for human CYP1B1 we have found no evidence for binding to mouse CYP1B1 . This data establishes CYP1B1 as an important contributor to activation of PAHs, particularly in extra hepatic tissues that are susceptible to cancer where CYP1B1 in contrast to CYP1A1 is constitutively expressed . Cancer, 1997 Dec 15, 80(12 Suppl), 2699 - 705 Pretargeting using peptide nucleic acid; Rusckowski M et al.; BACKGROUND: Pretargeting studies in animals and humans have usually involved (strept)avidin and biotin . Depending on the particular strategy, endogenous biotin can adversely influence localization when these molecules are used . METHODS: As an alternative to (strept)avidin and biotin, we have explored the use of a single-stranded peptide nucleic acid (PNA) bound to a protein administered first and followed by the complementary single-stranded PNA radiolabeled with 99mTc . Target localization of the PNA-bound protein in a mouse infection and a mouse tumor model occurred by passive diffusion while the radiolabeled complementary PNA localized by in vivo hybridization . The PNA-streptavidin was prepared by adding biotin-conjugated PNA to streptavidin; the complementary PNA, derivatized with a primary amine, was conjugated with acetyl S-protected NHS-MAG3 bifunctional chelator and radiolabeled with 99mTc . RESULTS: In both the infection and tumor mouse models, increased localization of radiolabel was achieved in animals receiving both injectates compared with control animals receiving only the radiolabeled PNA . In the infection model, the infected to normal thigh radioactivity ratio was 3.5 for the study animals compared with 1.7 for control animals (P = 0.0001) . In the tumor model, these values were 1.7 versus 1.2 (P = 0.003) . CONCLUSIONS: We conclude that PNA may be considered an alternative to (strept)avidin and biotin for pretargeting studies. J Clin Lab Anal, 1997, 11(6), 315 - 22 Reactivity of recombinant Treponema pallidum (r-Tp) antigens with anti-Tp antibodies in human syphilitic sera evaluated by ELISA; Fujimura K et al.; We evaluated the immunoreactivity of recombinant Treponema pallidum (r-Tp) antigens with human sera by indirect enzyme-linked immunoabsorbant assay (ELISA) . We expressed antigens with a molecular weight (MW) of 17KDa, 15KDa, 47KDa, and 42KDa, which are believed to be major immunoreactive membrane proteins of Tp cells . The expressed proteins were described by adding the prefix M, S, and G to the corresponding Tp antigens, namely, mature antigens, signal sequence containing antigens, and glutathione s-transferase (GST)-fused antigen in this report . A rather high expression occurred for M47 and S42 proteins in the Escherichia coli system, whereas for M15 and M17 proteins, a poor expression was observed . However, a fairly high expression occurred for G15 and G17 . Thus expressed proteins were purified by means of chromatographies to a level of > 95%, and the purified proteins were found to be reactive with TPHA positive serum by Western blotting (WB) . An ELISA performed with a serum of 1/1000 dilution using these purified antigens for coating on the solid phase showed that G17 antigen was more effective in detecting syphilis antibodies in human serum than M47, S42, and G15 . There was a good consistency between ELISA and TPHA, whereby the cutoff indexes (CI) on ELISA showed a correlation coefficient of 0.7276 in logarithmic TPHA titers. Brain Res Mol Brain Res, 1997 Oct 15, 50(1-2), 107 - 12 Ibuprofen: effect on inducible nitric oxide synthase; Stratman NC et al.; Treatment with ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDS) has been reported to decrease the incidence as well as slow down the progression of Alzheimer's disease . Understanding the mechanism of this therapeutic effect would provide a target for development of drugs which may be devoid of side effects observed with NSAIDs . In addition to inhibiting cyclooxygenase (COX), the NSAIDs have recently been shown to decrease inducible nitric oxide synthase (iNOS) activity . Ibuprofen and other NSAIDs had no direct effect on catalytic activity of iNOS, but decreased levels of iNOS mRNA . The mechanism of action of ibuprofen on reduction of iNOS activity has been further investigated in the present study using rat primary cerebellar glial cell cultures . In addition, the effect of ibuprofen on COX mRNA expression and prostaglandin formation was also studied . Glial cells treated with E . coli lipopolysaccharide (LPS) and interferony (INFgamma) for 16 h expressed iNOS and COX . Ibuprofen did not directly inhibit iNOS activity . However, when ibuprofen was incubated at the same time with LPS and INFgamma for 16 h, enzyme activity was reduced, with an IC50 of 0.76 mM . Ibuprofen concentration-dependently decreased iNOS mRNA levels, with an IC50 > 2 mM . Thus, there was no correlation between decrease in iNOS activity and reduction in iNOS mRNA levels . Ibuprofen decreased iNOS protein levels, as determined by Western blot, with an IC50 of 0.89 mM . The data suggest that the reduction in iNOS activity by ibuprofen is due to inhibition of post-transcriptional processing of this enzyme . Ibuprofen had no effect on constitutive COX (COX-1) or inducible COX (COX-2) mRNA expression . However, ibuprofen inhibited PGE2 formation with an IC50 of 0.86 mM . The anti-inflammatory actions of ibuprofen have been related to inhibition of COX and, subsequently, reducing prostaglandin formation . Since the potency of ibuprofen for inhibition of PGE2 formation and reduction in iNOS activity are similar, it is suggested that, at therapeutically effective doses, a decrease in iNOS activity may also occur in vivo . Therefore, reduction in iNOS protein levels in the brain may have a role in preserving the integrity of neurons in individuals susceptible to Alzheimer's disease. Mol Cell Endocrinol, 1997 Oct 20, 133(2), 177 - 82 FGF-2 antisense RNA encodes a nuclear protein with MutT-like antimutator activity; Li AW et al.; Bidirectional transcription of the basic fibroblast growth factor (FGF-2) gene gives rise to multiple polyadenylated sense mRNAs and a unique 1.5 kb antisense transcript (FGF-AS) which is complementary to the 3'-untranslated region of the FGF-2 mRNA . The rat FGF-AS cDNA encodes a novel 35 kDa nuclear protein (GFG) with homology to the MutT family of antimutator NTPases . Antibodies against the deduced amino acid sequence of GFG detected intense immunoreactivity in the nuclei of adult rat hepatocytes . Subcellular fractionation and Western blotting confirmed the presence of a 35 kDa immunoreactive protein in the nuclear fraction and, to a lesser extent, in the mitochondrial fractions of rat liver homogenates . Recombinant GFG suppressed the spontaneous mutation rate of MutT-deficient E . coli in a complementation assay . In-frame deletion of the 53 amino acids encompassing the MutT domain eliminated this activity, confirming the catalytic function of this region in the FGF antisense gene product . These findings demonstrate for the first time that the FGF-AS transcript encodes a functional nuclear protein with MutT-related enzymatic activity. Mol Cell Endocrinol, 1997 Oct 20, 133(2), 99 - 107 The full agonistic effect of recombinant 20 kDa human growth hormone (hGH) on CHO cells stably transfected with hGH receptor cDNA; Wada M et al.; The agonistic effect of the recombinant 20 kilodalton human GH (20K-hGH) with authentic primary structure was studied using Chinese hamster ovary (CHO) cells stably transfected with hGH receptor (hGHR) cDNA and was compared with that of 22K-hGH . The binding affinities (dissociation constants) of 20K- and 22K-hGH were identical (0.41 +/- 0.11 nM and 0.41 +/- 0.04 nM, respectively) . In addition, the two hGHs possessed the same potencies in activating the rat serine protease inhibitor (Spi) 2.1 gene promoter . 20K-hGH was similarly internalized as 22K-hGH but its internalization rate was a little slower than that of 22K-hGH . We also found that proliferation of CHO-hGHR cells stimulated by serum was remarkably inhibited by both hGHs to the same degree . In conclusion, both hGH isoforms exhibited the same binding affinities for hGHR and were potent enough to induce some hGHR-mediated cellular events . These suggest that 20K-hGH exerts a full agonistic activity for hGHR. Anat Embryol (Berl), 1997 Nov, 196(5), 363 - 82 Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex; Terashima T et al.; Adenoviral vectors have recently been recognized as highly efficient systems for gene delivery into various tissues . We show that a reporter gene introduced into nerve terminals via an adenovirus can be used to label cell bodies retrogradely and then label the axons and nerve terminals of the infected neurons anterogradely in vivo . We injected a replication-defective recombinant adenovirus carrying the E . coli beta-galactosidase gene (lacZ) into the cerebellar cortex of the adult mouse . The first evidence of retrograde labeling was obtained at 2 days after the infection when neurons in the pontine nuclei and the reticulotegmental nucleus of the pons weakly expressed beta-galactosidase, and at 3 days post-infection when neurons in all precerebellar nuclei, known to project to the cerebellar cortex, were strongly stained with X-gal in a Golgi-like manner . Anterograde transport of lacZ gene products was recognized at 3 days post-infection; beta-galactosidase-positive axons arose from somata or dendrites of retrogradely labeled neurons, passed through the middle or inferior cerebellar peduncles, and entered the cerebellum . Anterogradely labeled mossy terminals were recognized on the injection side at 8 days post-infection, and on the contralateral side at 14 days post-infection . Beta-galactosidase expression persisted for up to two months, with a decrease in the total number of labeled cells over time . We could not find any signs of anterograde or retrograde transsynaptic labeling in the nuclei synaptically linked to the cerebellar cortex at any time point after injection up to 58 days post-infection. J Med Chem, 1997 Dec 5, 40(25), 4013 - 8 Intensely cytotoxic anthracycline prodrugs: glucuronides; Bakina E et al.; We previously reported the synthesis of a series of doxorubicin analogue prodrugs that give rise to intensely cytotoxic metabolites in the presence of carboxylate esterases . We now report studies on structurally related beta-glucuronide prodrugs that are converted to similar potent metabolites in the presence of beta-glucuronidases . These prodrugs were prepared by reductive condensation of daunomycin or doxorubicin with methyl 1-O-{(1'RS)-1'-ethoxy-4'-oxobutyl}-2,3,4-tri-O-acetyl-beta-D- glucopyranosyluronate in the presence of sodium cyanoborohydride followed by base-mediated cleavage of the glucuronate protective groups . The doxorubicin derivatives were isolated in very low yield, most likely because of the inherent base lability of the parent aglycone . By contrast, fairly good yields of the more base-stable daunomycin analogues were obtained . The target daunomycin glucuronide, N-{(4"RS)-4"-ethoxy-4"-(sodium 1"'-O-beta-D-glucopyranuronate)butyl}daunorubicin (6a), had a half-life of 30 h when incubated at a concentration of 12 microM in aqueous 0.05 M phosphate buffer, pH 7.4, at 37 degrees C . Under identical conditions in the presence of 197 units/mumol of Escherichia coli beta-glucuronidase, 6a was hydrolyzed with a half-life of 1.7 h . The single metabolite observed was chromatographically identical with that formed from the hydrolysis of N-(4,4-diacetoxybut-1-yl)daunomycin by carboxylate esterases . 6a was approximately 10,000-fold more toxic to human A375 melanoma cells in the presence of E . coli beta-glucuronidase than in the absence of the enzyme . These findings indicate the therapeutic potential of anthracycline glucuronide prodrugs as independent entities or four use in conjunction with enzyme tissue-targeting strategies such as antibody-directed enzyme prodrug therapy (ADEPT) or gene-directed enzyme prodrug therapy (GDEPT). Nat Struct Biol, 1997 Dec, 4(12), 1039 - 46 A designed four helix bundle protein with native-like structure; Schafmeister CE et al.; A 108 amino acid protein was designed and constructed from a reduced alphabet of seven amino acids . The 2.9 A resolution X-ray crystal structure confirms that the protein is a four helix bundle, as it was designed to be . Hydrogen/deuterium exchange experiments reveal buried amide protons with protection factors in excess of 1 x 10(6) in the range characteristic of well protected protons in functional folded proteins (10(3)-10(8)) rather than protons in rapid exchange (0-10(2)) . The protein is monomeric at 1 mM, the concentration at which the exchange experiments were undertaken, indicating that the exchange factors are due to a unique stable tertiary structure fold, and not due to any higher order quaternary structure . Thermodynamic analysis provides an estimate of the free energy of folding of -9.3 kcal mole-1 at 25 degrees C, consistent with the free energy of folding derived from the protection factors of the most protected protons, indicating that global unfolding is required for exchange of the most protected protons. Nat Struct Biol, 1997 Dec, 4(12), 979 - 83 Dimerization of the UmuD' protein in solution and its implications for regulation of SOS mutagenesis; Ferentz AE et al.; NMR spectroscopy has been used to determine that the dimerization interface of UmuD' in solution is not the homodimer interface originally inferred from crystallographic data . Instead, it resembles an interface that had been hypothesized to be involved in filamentation. Anal Chem, 1997 Dec 1, 69(23), 4828 - 32 Ultrasensitive voltammetric detection of underivatized oligonucleotides and DNA; Singhal P et al.; Electrochemical detection of nucleotides, ssDNA, and dsDNA was accomplished by using sinusoidal voltammetric detection at copper microelectrodes . Generally, detection of these molecules utilizes the electroactive nature of adenine and guanine residues at most electrode surfaces . The detection approach used in this study is based on the electrocatalytic oxidation of sugars and amines at copper surfaces . All nucleotides and DNA molecules comprise a ribose sugar backbone and primary amines present on the different nucleobases . Consequently, the detection approach is universal to all types of nucleotides . As the number of sugar moieties increases with the length of an oligonucleotide, the detection sensitivity is enhanced for bigger oligonucleotides . Irreversible adsorption of these oligonucleotides and other biomacromolecules like dsDNA on the electrode surface was avoided with sinusoidal voltammetry since it is a scanning electrochemical technique . The sensitivity of the detection strategy is, however, still preserved due to the effective decoupling of the faradaic signal from the capacitive background currents in the frequency domain . The ssDNA and dsDNA were detected in the picomolar concentration range . The electrochemical signal due to dsDNA is actually higher than that due to ssDNA due to the larger number of easily accessible sugars on the outer perimeter of a dsDNA double helix compared to those on a ssDNA of the same size . This is in contrast to the existing electrochemical detections techniques based on the electroactivity of the nucleobase. Appl Environ Microbiol, 1997 Dec, 63(12), 4721 - 8 Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex; Li XL et al.; Two cDNAs encoding two cellulases, CelA and CelC, were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp . strain PC-2 constructed in Escherichia coli . Nucleotide sequencing revealed that the celA cDNA (1,558 bp) and celC cDNA (1,628 bp) had open reading frames encoding polypeptides of 459 (CelA) and 449 (CelC) amino acids, respectively . The two cDNAs were 76.9 and 67.7% identical at the nucleotide and amino acid levels, respectively . Analysis of the deduced amino acid sequences showed that starting from the N termini, both CelA and CelC had signal peptides, which were followed by noncatalytic repeated peptide domains (NCRPD) containing two repeated sequences of 33 to 40 amino acid residues functioning as docking domains . The NCRPDs and the catalytic domains were separated by linker sequences . The NCRPDs were homologous to those found in several hydrolases of anaerobic fungi, whereas the catalytic domains were homologous to the catalytic domains of fungal cellobiohydrolases and bacterial endoglucanases . The linker sequence of CelA contained predominantly glutamine and proline residues, while that of CelC contained mainly threonine residues . CelA and CelC did not have a typical cellulose binding domain (CBD) . CelA and CelC expressed in E . coli rapidly decreased the viscosity of carboxymethyl cellulose (CMC), indicating that there was endoglucanase activity . In addition, they produced cellobiose from CMC, acid-swollen cellulose, and cellotetraose, suggesting that they had cellobiohydrolase activity . The optimal activity conditions with CMC as the substrate were pH 4.3 to 6.8 and 50 degrees C for CelA and pH 4.6 to 7.0 and 40 degrees C for CelC . Despite the lack of a CBD, CelC displayed a high affinity for microcrystalline cellulose, whereas CelA did not. Appl Environ Microbiol, 1997 Dec, 63(12), 4671 - 8 Ribotyping to compare Fusobacterium necrophorum isolates from bovine liver abscesses, ruminal walls, and ruminal contents; Narayanan S et al.; Restriction fragment length polymorphism analysis of rRNA genes was employed to genetically compare Fusobacterium necrophorum subsp . necrophorum and F . necrophorum subsp . funduliforme isolates from multiple abscesses of the same liver and isolates from liver abscesses, the ruminal wall, and ruminal contents from the same animal . Four livers with multiple abscesses and samples of ruminal contents, ruminal walls, and liver abscesses were collected from 11 cattle at slaughter . F . necrophorum was isolated from all liver abscesses, nine ruminal walls, and six ruminal content samples . Chromosomal DNA of the isolates was extracted and single or double digested with restriction endonucleases (EcoRI, EcoRV, SalI, and HaeIII); then restriction fragments were hybridized with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli . EcoRI alone or in combination with EcoRV yielded the most discriminating ribopatterns for comparison . Within the subspecies multiple isolates from the same liver were indistinguishable based on the ribopattern obtained with EcoRI . The hybridization patterns of liver abscess isolates were concordant with those of the corresponding isolates from ruminal walls in eight of nine sets of samples . None of the six ruminal content isolates matched either the liver abscess isolates or the ruminal wall isolates . The genetic similarity between the isolates from liver abscesses and ruminal walls supports the hypothesis that F . necrophorum isolates of liver abscesses originate from the rumen. Appl Environ Microbiol, 1997 Dec, 63(12), 4651 - 6 Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes; Galkin A et al.; We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes . L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH . We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase . L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E . coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess) . Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E . coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH . Optically pure D enantiomers of glutamate and leucine were obtained. Oncol Res, 1997, 9(6-7), 313 - 25 Targeting gene therapy to cancer: a review; Dachs GU et al.; In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root . This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide . This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment . In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation . Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods . In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy . To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail . To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue . Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC) . Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements . Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing . Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia . We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail . Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply . In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized . Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue . This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy . However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target . We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors . The list of genes that have been considered for use in the treatment of cancer is extensive . It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop . Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes) . This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E . (ABSTRACT TRUNCATED) J Dairy Sci, 1997 Nov, 80(11), 2826 - 32 Concentrations of alpha-Tocopherol after intramammary infusion of Escherichia coli or lipopolysaccharide; Barrett JJ et al.; Fifteen Holstein cows were used in a trial involving intramammary challenge to determine the effects of acute clinical mastitis on the concentrations of alpha-tocopherol in milk and plasma and the concentrations of neutrophils in milk and blood . Cows were assigned to one of three experimental groups challenged by intramammary infusion of lipopolysaccharide, Escherichia coli, or sterile phosphate-buffered saline . All quarters infused with lipopolysaccharide or E . coli were diagnosed with clinical mastitis on d 1 and 2 after challenge . Acute inflammation caused by intramammary infusion of lipopolysaccharide or E . coli resulted in increased concentrations of alpha-tocopherol in milk in challenged quarters but had no effect on concentrations of alpha-tocopherol in plasma . Concentrations of alpha-tocopherol in milk and blood neutrophils did not differ among treatment groups . Concentrations of alpha-tocopherol did not differ between milk and blood neutrophils . Approximately 25% of the alpha-tocopherol in milk from glands with clinical mastitis was associated with neutrophils, and < 10% of the alpha-tocopherol in milk from nonmastitic glands was associated with neutrophils . A shift toward sources of alpha-tocopherol other than synthesized milk fat occurred during acute inflammation in the mammary gland. Horm Metab Res, 1997 Oct, 29(10), 524 - 9 Dietary regulation of the very low density lipoprotein receptor in mouse heart and fat; Kwok S et al.; The very low density lipoprotein (VLDL) receptor is a member of the LDL receptor family . As opposed to the LDL receptor, the VLDL receptor is expressed primarily in muscle and adipose tissue . Although the VLDL receptor is capable of binding lipoproteins, its functional role is still unclear . Previous studies found that VLDL receptor expression is unaffected by fasting in the rat . The current studies examined whether VLDL receptor expression is altered with fasting in the mouse . Balb/c mice were fasted for periods up to 48 hours, killed, hearts and epididymal fat obtained, and total membranes prepared . To detect the VLDL receptor a portion of the rat VLDL receptor was expressed as a bacterial fusion protein, purified and used to immunize rabbits . The antibodies raised specifically recognized intact VLDL receptor . When cardiac membranes were immunoblotted, VLDL receptor expression increased progressively with fasting, doubling at 36 hours . In contrast, VLDL receptor expression decreased progressively with fasting in membranes from epididymal fat, being reduced 70% by 48 hours . Thus, VLDL receptor expression appears to be regulated in mouse heart and fat by nutritional perturbation, supporting a potential role for the VLDL receptor in the delivery of triglycerides/fatty acids as fuel. RNA, 1997 Dec, 3(12), 1480 - 5 Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center; Bulygin KN et al.; mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs . Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used . Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes . Crosslinked nucleotides were identified by primer extension . In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked . Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible. RNA, 1997 Dec, 3(12), 1444 - 55 Identification of a novel, non-snRNP protein complex containing U1A protein; O'Connor JP et al.; Mouse monoclonal antibodies (MAbs) were generated against Escherichia coli-produced U1snRNP-A (U1A) protein . U1A-specific MAbs as well as MAbs that reacted with both U1A and U2snRNP-B" (U2B") were isolated . MAb 12E12 was unique among the characterized MAbs because it failed to immunoprecipitate U1A protein produced by in vitro transcription and translation using rabbit reticulocyte lysates . However, when U1A protein was made using a wheat germ extract, MAb 12E12 could immunoprecipitate U1A quite readily, as did the other MAbs . These data suggest that the MAb 12E12 epitope is masked when U1A is prepared in reticulocyte lysate . Further studies showed that MAb 12E12 recognizes an epitope that is masked when U1A protein is bound to U1 RNA . The unique nature of MAb 12E12 was used to demonstrate that U1A could be immunoprecipitated from whole-cell extracts in a form that was free of U1 RNA and other snRNP components . However, this snRNP-free U1A (SF-A) was found to co-immunoprecipitate with a unique set of non-snRNP proteins . In order to confirm that U1A exists in at least two distinct complexes (snRNP bound and snRNP free), {35S}-labeled nucleoplasmic extracts were analyzed by sucrose density gradient fractionation and immunoprecipitation . MAb 12E12 specifically immunoprecipitated SF-A, which migrated in a novel non-snRNP complex . Specifically, proteins of approximately 58, 59, 63, 65, and 105 kDa co-sedimented and co-immunoprecipitated with SF-A . Our data show that a significant portion of the cellular U1A (at least 3% or approximately 30,000 molecules) exists in the nucleoplasm in one or more novel complexes . Our previous studies have demonstrated an effect of purified U1A on polyadenylation of pre-mRNAs and, consistent with this finding, purified antibodies to SF-A significantly diminish polyadenylation in vitro. Vet Parasitol, 1997 Oct, 72(2), 185 - 99 Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes; Brayton KA et al.; The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools . We have developed an immunoaffinity chromatographic method to purify C . ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA . This DNA has been used to construct what we believe to be the first fully representative C . ruminantium expression library . A clone containing the complete Cowdria map1 gene has been isolated and sequenced . This gene has been expressed in E . coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms . Parts of three other Cowdria genes have also been isolated and sequenced. Bioconjug Chem, 1997 Nov-Dec, 8(6), 878 - 84 Generation and characterization of an anti-CD19 single-chain Fv immunotoxin composed of C-terminal disulfide-linked dgRTA; Wang D et al.; Our laboratory utilized two methods to produce the anti-CD19 immunotoxin containing a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA) . The first method produced the recombinant protein FVS191CDRTA from a fusing gene containing sequences encoding FVS191, catheptsin D proteinase digestion site (CD), and RTA . FVS191CDRTA did not show CD19 antigen binding and cytotoxic activity . The second method generated a disulfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an additional C-terminal cysteine, and a deglycosylated RTA (dgRTA) . The formation of FVS191cys-dgRTA is efficient; up to 70% of the proteins participating in the reaction had formed FVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1 . A competitive ELISA assay indicated that FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities . The protein synthesis inhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent than the intact antibody-conjugated B43-dgRTA, which had an IC50 = 2 x 10(-11) M . 125I-Labeled FVS191 and 125I-labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demonstrated by internalization studies; this result indicates that cross-linking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA is not due to the monovalent binding of CD19 by FVS191cys-dgRTA . Our study with anti-CD19 scFv immunotoxin indicates that the formation of a disulfide-linked scFv immunotoxin is an alternative to the recombinant method of producing scFv immunotoxin. Bioconjug Chem, 1997 Nov-Dec, 8(6), 798 - 812 Role of the central metal ion and ligand charge in the DNA binding and modification by metallosalen complexes; Mandal SS et al.; Several metal complexes of three different functionalized salen derivatives have been synthesized . The salens differ in terms of the electrostatic character and the location of the charges . The interactions of such complexes with DNA were first investigated in detail by UV-vis absorption titrimetry . It appears that the DNA binding by most of these compounds is primarily due to a combination of electrostatic and other modes of interactions . The melting temperatures of DNA in the presence of various metal complexes were higher than that of the pure DNA . The presence of additional charge on the central metal ion core in the complex, however, alters the nature of binding . Bis-cationic salen complexes containing central Ni(II) or Mn(III) were found to induce DNA strand scission, especially in the presence of co-oxidant as revealed by plasmid DNA cleavage assay and also on the basis of the autoradiogram obtained from their respective high-resolution sequencing gels . Modest base selectivity was observed in the DNA cleavage reactions . Comparisons of the linearized and supercoiled forms of DNA in the metal complex-mediated cleavage reactions reveal that the supercoiled forms are more susceptible to DNA scission . Under suitable conditions, the DNA cleavage reactions can be induced either by preformed metal complexes or by in situ complexation of the ligand in the presence of the appropriate metal ion . Also revealed was the fact that the analogous complexes containing Cu(II) or Cr(III) did not effect any DNA strand scission under comparable conditions . Salens with pendant negative charges on either side of the precursor salicylaldehyde or ethylenediamine fragments did not bind with DNA . Similarly, metallosalen complexes with net anionic character also failed to induce any DNA modification activities. Biochem Cell Biol, 1997, 75(3), 199 - 205 Structural characterization of the serotype O:5 O-polysaccharide antigen of the lipopolysaccharide of Escherichia coli O:5; MacLean LL et al.; The structure of the antigenic O-polysaccharide component of the smooth lipopolysaccharide produced by Escherichia coli serotype O:5 was investigated by composition, methylation, and periodate oxidation methods, and by 1D and 2D nuclear magnetic resonance spectroscopy . The antigenic O-chain was determined to be a high molecular weight polysaccharide composed of repeating tetrasaccharide units containing 2-acetamido-2-deoxy-D-galactose, D-galactose, D-ribose, and 3-acetamido-3,6-dideoxy-D-glucose and having the structure -{-4)-beta-D-Galp-(1-3)-alpha-D-GalpNAc-(1-4)-beta-D-Quinp3NAc-(1- 3)-beta-D-Ribf-(1-}-. Osteoarthritis Cartilage, 1997 Jul, 5(4), 275 - 82 Transplantation of adenovirally transduced allogeneic chondrocytes into articular cartilage defects in vivo; Baragi VM et al.; Gene transfer to chondrocytes followed by intra-articular transplantation may allow for functional modulation of chondrocyte biology and enhanced repair of damaged articular cartilage . We chose to examine the loss of chondrocytes transduced with a recombinant adenovirus containing the gene for Escherichia coli beta-galactosidase (Ad.RSVntlacZ), followed by transplantation into deep and shallow articular cartilage defects using New Zealand White rabbits as an animal model . A type I collagen matrix was used as a carrier for the growth of the transduced chondrocytes and to retain the cells within the surgically created articular defects . Histochemical analysis of matrices recovered from the animals 1, 3 and 10 days after implantation showed the continued loss of lacZ positive chondrocytes . The number of cells recovered from the matrices was also compared with the initial innoculum of transduced cells present within the matrices at the time of implantation . The greatest loss of transduced cells was observed in the first 24 h after implantation . The numbers of transduced cells present within the matrices were relatively constant between 1 and 3 days postimplantation, but had progressively declined by 10 days postimplantation . These results suggest that transduction of chondrocytes followed by intra-articular transplantation in this rabbit model may enable us to examine the biological effects of focal transgenic overexpression of proteins involved in cartilage homeostasis and repair. Biosci Biotechnol Biochem, 1997 Nov, 61(11), 1955 - 6 Synthesis of asymmetrically labeled sucrose by a recombinant sucrose synthase; Nakai T et al.; About 80% of radioactivity was recovered in asymmetrically labeled sucrose from UDP-{14C}glucose or {14C}fructose with recombinant mung bean sucrose synthase expressed in Escherichia coli harboring pEB-01 . This high recovery is due to the fact that the enzyme conserving the activity of sucrose synthase has a similar affinity for UDP-glucose and fructose to an intact enzyme from the mung bean, but a lower affinity for sucrose. Biosci Biotechnol Biochem, 1997 Nov, 61(11), 1949 - 52 Human placental fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase: its isozymic form, expression and characterization; Sakakibara R et al.; The nucleotide sequence of 1981 bp cDNA containing the entire coding region of a human placental fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase was determined . The sequence encodes 469 amino acids and, based on homology to the rat testis enzyme, appears to be the testis-type isozyme expressed in placenta . The enzyme was expressed in Escherichia coli BL21 (DE3) by using a T7 RNA polymerase-based expression system and purified to homogeneity . The expressed enzyme was bifunctional with specific activities of 75 and 80 mU/mg of kinase and phosphatase, respectively . Kinetic parameters of the expressed enzyme are similar to those of the rat testis enzyme. Arch Surg, 1997 Dec, 132(12), 1342 - 7 Endotoxin-induced macrophage gene expression depends on platelet-activating factor; Lo CJ et al.; BACKGROUND: The development of multiple organ failure in septic patients is due to a systemic inflammation orchestrated by macrophages (Mphi) . Elucidation and control of the mechanism involved in Mphi activation in sepsis is crucial to improving survival . An early event of Mphi activation involves the hydrolysis of membrane phospholipid by phospholipase A2 (PLA2) and subsequent generation of platelet-activating factor (PAF) . OBJECTIVE: We designed this study to test the hypothesis that Mphi gene expression depends on PAF . DESIGN: Rabbit alveolar Mphi were obtained by bronchoalveolar lavage and were stimulated with 10 ng/mL of Escherichia coli endotoxin lipopolysaccharide (LPS), PAF (1 micromol/L), LPS+/-CV3988 (10 micromol/L), a PAF receptor antagonist, or LPS+/-PLA2 inhibitors: AACOCF3 (50 micromol/L) or manoalide (10 micromol/L) . After 4 hours of incubation, Mphi tumor necrosis factor (TNF) messenger RNA (mRNA) expression was assessed by Northern blot analyses . The TNF production in the Mphi supernatant was measured by L929 bioassays . RESULTS: The LPS-stimulated Mphi expressed increased levels of TNF mRNA and produced an enormous amount of TNF . CV3988, a PAF antagonist, inhibited LPS-induced TNF mRNA . Furthermore, inhibiting PAF production with AACOCF3, or manoalide, also inhibited LPS-induced Mphi TNF mRNA expression . The effect of PAF depends on changes in intracellular calcium concentration . Inhibitors of calcium flux attenuated the PAF effects on LPS-stimulated Mphi . CONCLUSIONS: Our data suggest that LPS-induced Mphi gene expression is mediated by PAF . It is likely that modulation of PAF production or activity may be beneficial in down-regulating the overactivity of Mphi in sepsis. Microbiol Immunol, 1997, 41(10), 841 - 5 Immune response to neutralizing epitope on human cytomegalovirus gylcoprotein B in Japanese: correlation of serologic response with HLA-type; Wada K et al.; Antigenic domain 1 (AD-1), located between amino acids 608 and 625 of human cytomegalovirus (CMV) gB protein, is the major domain recognized by neutralizing antibodies . Amino acids 552 to 630 are essential for the binding of neutralizing antibodies . We developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against a fusion protein containing amino acid residues 549 to 644 of the gB polypeptide and maltose binding protein (MBP) . Of 180 seropositive samples, 106 (58.9%) showed positive immuno-reactivity against the fusion protein . None of the seronegative samples reacted with the fusion protein . Among 57 seropositive individuals typed for HLA, subjects with HLA-DR9 had a higher positive rate against the fusion protein (13/14=92.9%) than those without HLA-DR9 (25/43=58.1%) . In addition, subjects with HLA-DR15 had a lower positive rate against the fusion protein (7/16=43.3%) than those without HLA-DR15 (31/41=75.6%) . Mean OD values of HLA-DR15-positive individuals were significantly lower than those of HLA-DR15-negative individuals . Thus, among CMV-infected individuals, HLA-DR9 may be associated with responders for neutralizing antibodies and HLA-DR15 may be associated with non/low-responders. Microbiol Immunol, 1997, 41(10), 805 - 8 Cloning and sequence analysis of another Shiga-like toxin IIe variant gene (slt-IIera) from an Escherichia coli R107 strain isolated from rabbit; Kim SH et al.; An Escherichia coli R107 strain (O26 serotype) producing a Shiga-like toxin IIe variant (SLT-IIera) was isolated from the mesenteric lymph node of a freshly dead rabbit carcass . The entire structural gene for this SLT-IIera was cloned from chromosomal DNA by PCR using primers based on previously published slt-IIe sequences . Nucleotide sequence analysis indicated that the slt-IIera gene was very similar to slt-IIe (formerly called slt-IIv) from E . coli strains S1191 and 412; five and one nucleotide changes were detected in A and B subunits, respectively, which resulted in changes in amino acid sequences of the corresponding subunits by three and one residues . Recombinant SLT-IIera and SLT-IIe produced using an E . coli host-vector system showed similar cytotoxicity, suggesting that the variations in the structural gene of SLT-IIera have no significant effect on cytotoxic level. Biol Trace Elem Res, 1997 Sep, 58(3), 169 - 76 Effect of dietary selenium on the response of stressed and unstressed chickens to Escherichia coli challenge and antigen; Larsen CT et al.; Selenium was added to the feed of White Leghorn type chickens 1 day prior to challenge with either Escherichia coli or sheep erythrocyte antigen . The incidence of death or lesions was reduced from 86% to 21% at the optimal dose of selenium (0.4 mg/kg resulting in feed concentration of 0.45 mg/kg) . After the chickens were stressed by chilling, selenium was ineffective against E . coli . Dietary additions of selenium between 0.1 and 0.8 mg/kg resulted in an antibody titer increase from 2.2 to 3.9 to the log2 against sheep erythrocytes (SRBC) . Following chilling, antibody titer response was reduced from 4.9 to 2.4 to the log2 . This titer reduction could be prevented with dietary additions of selenium between 0.1 and 1.2 mg/kg . The effects of a nitrofuran and selenium were additive against E . coli challenge infection. Mutat Res, 1997 Nov 19, 381(1), 111 - 5 Involvement of recA and recF in the induced precise excision of Tn10 in Escherichia coli; Chan A et al.; It has been shown that the increased frequency of precise excision of Tn10 observed after UV or mitomycin C (MMC) treatment or with uvrD- mutants is recA-dependent . Previous work has also shown that expression of SOS genes is required for UV- or MMC-induced Tn10 precise excision . In order to determine if the increased excision of Tn10 in uvrD- mutants requires only expression of recA, or expression of other SOS genes, or both, we studied the precise excision of Tn10 in lexA3 (Ind-, SOS non-inducible) and lexA3 recAo98 (operator constitutive recA) mutants . The results of these experiments indicate that the induced excision of Tn10 in the uvrD- null mutant depends on the expression of recA rather than on any of the other genes repressed by LexA . The effect of a null recF mutation on the excision of Tn10 in a uvrD- mutant was also investigated and found to abolish the increased frequencies of this process . Similarly, the recF mutation was found to decrease markedly the increased precise excision of Tn10 induced by MMC in a uvrD+ isogenic strain . These observations indicate that recA and recF are involved in the increased frequencies of Tn10 excision exhibited by uvrD- mutants or after MMC treatment . It remains to be determined whether these two genes participate in these two induction processes in the same biochemical pathways. J Am Soc Nephrol, 1997 Dec, 8(12), 1877 - 88 Verocytotoxin inhibits mitogenesis and protein synthesis in purified human glomerular mesangial cells without affecting cell viability: evidence for two distinct mechanisms; Van Setten PA et al.; Acute renal failure is one of the hallmarks of the hemolytic uremic syndrome (HUS) . Infection with a verocytotoxin (VT)- or Shiga-like toxin (SLT)-producing Escherichia coli has been strongly implicated in the etiology of the epidemic form of HUS . The functional receptor for these closely related toxins appears to be a glycosphingolipid, globotriaosylceramide (Gb3) . Endothelial damage in the glomeruli and arterioles of the kidney induced by VT is believed to play a crucial role in the pathogenesis of HUS . However, little information is available regarding the effects of VT on mesangial cells, which also play an important role in glomerular function . In this study, the effects of VT on human mesangial cells in vitro were investigated . Mesangial cells were enriched by collecting hillock-shaped outgrowths derived from adult human glomeruli and subsequently purified by elimination of contaminating epithelial cells by immunoseparation with ulex europaeus lectin-I (UEA-I)-coated dynabeads . The obtained and subcultured mesangial cell populations were >98% pure . Their mesangial nature was established by the presence of a-smooth muscle cell actin in highly confluent cultures and the absence of cytokeratin or platelet/endothelial cell adhesion molecule-1 . Mesangial cells bound VT to bands of Gb3 and a closely related glycolipid, which is similar to a glycolipid involved in the VT-dependent cytokine production in monocytes . VT did not induce the release of cytokines or chemokines in mesangial cells . In VT-susceptible cells, binding of VT to Gb3 causes cell death by the inhibition of protein synthesis . Although protein synthesis was inhibited in mesangial cells, all cells remained viable, both under basal and tumor necrosis factor-alpha-stimulated conditions . However, the marked reduction in protein synthesis may impair a proper response of the cells in conditions of increased demand of newly synthesized proteins . Furthermore, VT markedly inhibited DNA synthesis and proliferation of mesangial cells . The inhibition of mitogenesis was also found with the B-subunit of VT-1 alone, albeit to a lesser extent, without a significant effect on protein synthesis . Because the inhibition of protein synthesis involves the A-subunit, this suggests that two distinct mechanisms contribute to the effects of VT on protein synthesis and mitogenesis . Intracellular routing of VT (A- and B-subunits) may vary between cell types and result in differential effects on human mesangial cells when compared with other cell types. Mol Microbiol, 1997 Nov, 26(3), 597 - 606 Repair by recombination of DNA containing a palindromic sequence; Leach DR et al.; We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence . Wild-type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable . The dependence on homologous recombination for cell viability is overcome in sbcC mutants . Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants . Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination . The repair is conservative and the palindrome is retained in the repaired chromosome . We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold. Mol Microbiol, 1997 Nov, 26(3), 569 - 80 Blocking rolling circle replication with a UV lesion creates a deletion hotspot; Seigneur M et al.; UV light irradiation increases genetic instability by causing mutations and deletions . The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids . Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide . A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed . In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots . These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate . They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion . Furthermore, UV-induced deletions occurred independently of pBR322 replication . We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone . We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase. Mol Microbiol, 1997 Nov, 26(3), 557 - 67 uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway; Bierne H et al.; It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli . Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant . In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process . Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene . Two of the mutations were identified as opal mutations at codons 130 and 438 . A uvrD::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants . The uvrD-mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes . We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway . In uvrD+ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation . This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein . Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion . However, this stimulation is RecA independent, as is deletion in a wild-type strain . We propose that the presence of an excess of the UvrD helicase favours replication slippage . This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion. Mol Microbiol, 1997 Nov, 26(3), 519 - 30 FIS modulates growth phase-dependent topological transitions of DNA in Escherichia coli; Schneider R et al.; The Escherichia coli DNA-binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site-specific inversion of DNA and transcriptional activation of stable RNA promoters . In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes . We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA . However, FIS also binds and bends DNA at many non-specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E . coli chromosome . Here, we show that fis affects growth phase-specific alterations in the supercoiling level of DNA . Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift-up . In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state . Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth. Mol Microbiol, 1997 Nov, 26(3), 493 - 504 Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA; Soderbom F et al.; The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA . Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number . We show here that PcnB (PAPl-a poly(A)polymerase of Escherichia coli) is such a factor . Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased . We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA . These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment . We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent. Mol Microbiol, 1997 Nov, 26(3), 417 - 22 The Escherichia coli genome sequence: the end of an era or the start of the FUN? Hinton JC. Our dream of determining the entire Escherichia coli K12 genome sequence has been realized . This calls for new approaches for the analysis of gene expression and function in biology's best-understood organism . Comparison of the E . coli genome sequence with others will provide important taxonomic insights and have implications for the study of bacterial virulence . Approximately 20% of E . coli genes have been designated FUN genes, because they have no known function or homologies to sequence databases . FUN genes promise to have an exciting impact on bacterial research . The post-genome era requires novel strategies that address gene regulation at the level of the entire cell . These strategies need to supersede the reductionist approach to genetic analysis . Only then will the genome sequence lead us to an understanding of how a bacterial cell really works. Acta Physiol Scand, 1997 Nov, 161(3), 311 - 5 Effect of inhaled nitric oxide on endotoxin-induced hypoxaemia in rabbits; Heller H et al.; In five mechanically ventilated rabbits, we studied the property of inhaled nitric oxide in helping to treat hypoxaemia which was induced by intravenous endotoxin (Escherichia coli-derived lipopolysaccharide, serotype 0111: B4) . We u