Curr Opin Mol Ther, 2000 Aug, 2(4), 433 - 40 Developments in suicide genes for preclinical and clinical applications; Spencer DM; The graduation of gene therapy from unfulfilled dreams to conventional therapy for genetic and acquired disorders will require a mastery of multiple disparate components including gene delivery vectors, regulated tissue-specific gene expression, control of immunity and manipulation of cell viability . Improvements in suicide genes have opened up a whole new treatment modality for treating hyperproliferative disorders and for designing animal models for disease . Along with herpes simplex virus-1 thymidine kinase, a host of additional suicide genes have been developed . A critical comparison of these will follow along with progress in utilizing these reagents for therapeutic benefits. J Eukaryot Microbiol, 2001 Jan-Feb, 48(1), 11 - 6 Encystation in Acanthamoeba castellanii: development of biocide resistance; Lloyd D et al.; Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp . superb experimental systems for studies of cell biology and differentiation . More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites . Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli . Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment . For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process . After two hours, resistance to HCl was apparent . Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media . Chlorhexidine diacetate resistance was observed at between 24 and 36 h . The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated. Eur J Biochem, 2001 Mar, 268(6), 1861 - 8 Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase . A versatile tool to rate inhibitor effects? Ullrich A, Knecht W, Fries M, Loffler M. Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone . Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase . Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli . These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane . By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1 . Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein . The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes . The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species . The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors . Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal . Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action. Eur J Biochem, 2001 Mar, 268(6), 1794 - 801 Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue . Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase; De Vendittis E et al.; The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained . The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme . The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD {Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E . & Bocchini, V . (1999) J . Mol . Biol . 286, 189--205} . HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH(2)SO(2)F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH(2)SO(2)F . Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41 . PhCH(2)SO(2)F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased . The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD . These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD. Eur J Biochem, 2001 Mar, 268(6), 1739 - 48 Structural and functional properties of Escherichia coli-derived nucleoplasmin . A comparative study of recombinant and natural proteins; Hierro A et al.; Fourier transform infrared spectroscopy, circular dichroism and prediction techniques have been used to investigate the conformational properties of nucleoplasmin isolated from oocytes and eggs of Xenopus . laevis and overexpressed in Escherichia coli . A simple and fast method allows purification of recombinant nucleoplasmin free of truncated and/or aggregated forms, and therefore provides a suitable sample to carry out the structural and functional comparison between these proteins . The secondary structure of the three proteins estimated from both spectroscopic techniques was very similar, and was found to be 31--33% loops, 27--34% beta structure, 22--26% turns and 9-14% alpha helix . Prediction studies, in good agreement with experimental data, also suggest that beta structure is the major regular conformation, and that loops and turns are the most abundant conformational features within the secondary structure of nucleoplasmin . Furthermore, the spectroscopic characterization of a truncated version of the protein, lacking 80 residues at the C-terminus, and the prediction data indicate that the secondary structure elements of the protein are segregated into two regions . The N-terminal fragment (comprising residues 1--120) which holds all the putative beta strands, and the solvent-exposed C-terminal region, that is suggested to be enriched in turn and loop structures . The phosphate/protein monomer molar ratios, obtained from chemical analysis and mass spectrometry, are 0, 3 and 7--10 for recombinant, oocyte and egg nucleoplasmin, respectively . Phosphorylation does not significantly affect the secondary structure of the protein, but clearly modulates its ability to decondense sperm nuclei and to remove basic proteins from DNA. Eur J Biochem, 2001 Mar, 268(6), 1640 - 5 Contributions of the substrate-binding arginine residues to maleate-induced closure of the active site of Escherichia coli aspartate aminotransferase; Matharu A et al.; Crystallography shows that aspartate aminotransferase binds dicarboxylate substrate analogues by bonds to Arg292 and Arg386, respectively {Jager, J, Moser, M . Sauder, U . & Jansonius, J . N . (1994) J . Mol . Biol., 239, 285-305} . The contribution of each interaction to the conformational change that the enzyme undergoes when it binds ligands via these residues, is assessed by probing mutant forms of the enzyme lacking either or both arginines . The probes used are NaH(3)BCN which reduces the cofactor imine, the reactive substrate analogue, cysteine sulfinate and proteolysis by trypsin . The unreactive substrate analogue, maleate, is used to induce closure . Each single mutant reacted only 2.5-fold more slowly with NaH(3)BCN than the wild-type indicating that charge repulsion by the arginines contributes little to maintaining the open conformation . Maleate lowered the rate of reduction of the wild-type enzyme more than 300-fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue to bridge the arginines precisely makes the major contribution to closure . The R292L mutant reacted 20 times more rapidly with cysteine sulfinate than R386L but 5 x 10(4) times more slowly than the wild-type enzyme, consistent with the proposal that enzyme's catalytic abilities are not developed unless closure is induced by bridging of the arginines . Proteolysis of the mutants with trypsin showed that, in the wild-type enzyme, the bonds most susceptible to trypsin are those contributed by Arg292 and Arg386 . Proteolysis of the next most susceptible bond, at Arg25 in the double mutant, was protected by maleate demonstrating the presence of an additional site on the enzyme for binding dicarboxylates. FEBS Lett, 2001 Mar 9, 492(1-2), 66 - 72 Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters; Roerig P et al.; The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane . Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy . Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions . Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP . Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity . Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity . Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70 . The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter. FEBS Lett, 2001 Mar 9, 492(1-2), 54 - 7 Pigment conformation and pigment-protein interactions in the reconstituted Lhcb4 antenna protein; Pascal A et al.; Resonance Raman spectra of the native Lhcb4 antenna protein are compared with those of a recombinant protein prepared by in vitro refolding of its polypeptide, over-expressed in Escherichia coli, with added pigments {Giuffra et al . (1996) Eur . J . Biochem . 238, 112-120} . The results indicate that the native pigment conformation is reproduced almost perfectly in the reconstituted protein, with only small differences which are attributed to a slight shift in the Soret absorption peak of two or more chlorophylls . This procedure therefore represents a model system for the investigation of site-directed mutant LHC proteins, which are otherwise very difficult to obtain. Biochim Biophys Acta, 2001 May 1, 1505(1), 144 - 57 Na(+)/H(+) antiporters; Padan E et al.; Na(+)/H(+) antiporters are membrane proteins that play a major role in pH and Na(+) homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants . The emerging genomic sequence projects already have started to reveal that the Na(+)/H(+) antiporters cluster in several families . Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na(+)/H(+) antiporter of Escherichia coli . This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes . It has recently been crystallized in both 3D as well as 2D crystals . The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 A resolution was obtained and a 3D map was reconstructed . NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing . This is the first insight into the structure of a polytopic membrane protein . Many Na(+)/H(+) antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis . The molecular mechanism underlying this pH sensitivity has been studied in NhaA . Amino acid residues involved in the pH response have been identified . Conformational changes transducing the pH change into a change in activity were found in loop VIII-IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively . Regulation by pH of the eukaryotic Na(+)/H(+) antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am . J . Physiol . 269 (1995) G1-G11) . The transcription of NhaA has been shown to be regulated by a novel Na(+)-specific regulatory network . It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters. Toxicol Sci, 2001 Apr, 60(2), 356 - 62 Effects of ozone exposure on nuclear factor-kappaB activation and tumor necrosis factor-alpha expression in human nasal epithelial cells; Nichols BG et al.; In this study we investigated a possible mechanism of the human airway inflammatory response to inhaled ozone (O(3)) . Cultures of human nasal epithelial (HNE) cells, initiated from excised nasal turbinates and grown on collagen-coated Transwell tissue culture inserts, were exposed to 120, 240, or 500 ppb O(3) for 3 h . An electron spin resonance (ESR) signal that changed with time suggested free radical production in HNE cells exposed to O(3) . Nuclear protein extracts were analyzed for the activated transcription factor NF-kappaB by electrophoretic mobility-shift assay (EMSA), and showed a small dose-response activation of NF-kappaB that coincided with O(3)-induced free radical production . Basal media were analyzed for the presence of tumor necrosis factor-alpha (TNF-alpha) using the enzyme-linked immunosorbent assay (ELISA) . In cultures exposed to 120 ppb O(3), the mean TNF-alpha concentration was not significantly different from those exposed to air . However, exposure to 240 and 500 ppb O(3) significantly increased mean TNF-alpha expression, relative to controls, 16 h after exposure . These results support the hypothesis that the human airway epithelium plays a role in directing the inflammatory response to inhaled O(3) via free radical-mediated NF-kappaB activation. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3567 - 70 A method for soluble overexpression of the alpha7 nicotinic acetylcholine receptor extracellular domain; Fischer M et al.; We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor . The approach was to fuse the alpha7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli . An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the alpha7 ECD . This protein is soluble upon expression in bacteria and is purified by affinity chromatography . It binds the competitive nicotinic antagonist alpha-bungarotoxin with 2.5 microM affinity and displays a CD spectrum corresponding to a folded protein . The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3471 - 6 Epub 2001 Feb 27. The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal; Lee HC et al.; Previous studies have demonstrated that presecretory proteins such as maltose binding protein (MBP) and outer membrane protein A (OmpA) are targeted to the Escherichia coli inner membrane by the molecular chaperone SecB, but that integral membrane proteins are targeted by the signal recognition particle (SRP) . In vitro studies have suggested that trigger factor binds to a sequence near the N terminus of the mature region of OmpA and shunts the protein into the SecB pathway by blocking an interaction between SRP and the signal peptide . By contrast, we have found that the targeting pathway of a protein under physiological conditions is dictated by the composition of its targeting signal . Replacement of the MBP or OmpA signal peptide with the first transmembrane segment of AcrB abolished the dependence on SecB for transport and rerouted both proteins into the SRP targeting pathway . More modest alterations of the MBP signal peptide that simply increase its hydrophobicity also promoted SRP binding . Furthermore, we obtained evidence that SRP has a low affinity for typical signal peptides in vivo . These results imply that different classes of E . coli proteins are targeted by distinct pathways because bacterial SRP binds to a more restricted range of targeting signals than its eukaryotic counterpart. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3304 - 9 Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein; Kim SH et al.; IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer . The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule . In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage . Mature IL-18 generated by factor Xa cleavage was fully active . Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants . Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18 . A double mutant, E42A plus K89A, exhibited 4-fold greater activity . Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18 . The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18 . The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3288 - 93 Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography; Altamirano MM et al.; CD1 is an MHC class I-like antigen-presenting molecule consisting of a heavy chain and beta(2)-microglobulin light chain . The in vitro refolding of synthetic MHC class I molecules has always required the presence of ligand . We report here the use of a folding method using an immobilized chaperone fragment, a protein disulphide isomerase, and a peptidyl-prolyl cis-trans isomerase (oxidative refolding chromatography) for the fast and efficient assembly of ligand-free and ligand-associated CD1a and CD1b, starting with material synthesized in Escherichia coli . The results suggest that "empty" MHC class I-like molecules can assemble and remain stable at physiological temperatures in the absence of ligand . The use of oxidative refolding chromatography thus is extended to encompass complex multisubunit proteins and specifically to members of the extensive, functionally diverse and important immunoglobulin supergene family of proteins, including those for which a ligand has yet to be identified. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3092 - 7 Reverse engineering the (beta/alpha )8 barrel fold; Silverman JA et al.; The (beta/alpha)(8) barrel is the most commonly occurring fold among protein catalysts . To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (beta/alpha)(8) barrel enzyme triosephosphate isomerase . Using combinatorial mutagenesis and functional selection, we find that turn sequences, alpha-helix capping and stop motifs, and residues that pack the interface between beta-strands and alpha-helices are highly mutable . Conversely, any mutation of residues in the central core of the beta-barrel, beta-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity . Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli . At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity . Consequently, it seems likely that (beta/alpha)(8) barrel structures can be encoded with a subset of the 20 amino acids . Such simplification would greatly decrease the computational burden of (beta/alpha)(8) barrel design. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3080 - 5 Protein phosphatase 1 regulation by inhibitors and targeting subunits; Watanabe T et al.; Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeting subunits has been previously studied through the use of recombinant protein expressed in Escherichia coli . This preparation is limited by several key differences in its properties compared with native PP1 . In the present study, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus . Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting subunits, and failure to dephosphorylate a phosphotyrosine-containing substrate or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, M(r) 32,000) . Mutations at Y272 in the beta12/beta13 loop resulted in a loss of activity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2 . Mutations of Y272 also increased the relative activity toward a phosphotyrosine-containing substrate or phospho-DARPP-32 . Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibitor-2, but one mutant (E252A:D253A:E256R) exhibited an increased K(m) for phosphorylase a . Several PP1/PP2A chimeras were prepared in which C-terminal sequences of PP2A were substituted into PP1 . Replacement of residues 274-330 of PP1 with the corresponding region of PP2A resulted in a large loss of sensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclear targeting subunit (PNUTS) . More limited alterations in residues in beta12, beta13, and beta14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2. Comp Immunol Microbiol Infect Dis, 2001 Apr, 24(2), 135 - 42 Simultaneous isolation of verotoxin-producing strains of Escherichia coli O128:H2 and viruses in gastroenteritis outbreaks; Bettelheim KA et al.; Three outbreaks of gastroenteritis from which the Verotoxin producing Escherichia coli serotype O128:H2 was isolated are reported . In addition Norwalk-like viruses were detected in patients from two of the outbreaks and astrovirus in the third outbreak . While it cannot be specifically determined which of these agents played the major role in these outbreaks, the findings suggest that the viral agents need to be considered in investigations of gastroenteritis outbreaks, regardless of whether bacterial enteropathogens have also been isolated . This study points to a strong need to investigate gastroenteritis outbreaks for both bacterial and viral agents and to review in detail the asymptomatic carriage rate of Verotoxin-producing bacteria and gastroenteritis-associated viral agents; these areas of public health significance have been largely neglected. Crit Care Med, 2001 Feb, 29(2), 374 - 9 Methylene blue reduces lung fluid filtration during the early phase of endotoxemia in awake sheep; Evgenov OV et al.; OBJECTIVE: To determine whether methylene blue (MB), an inhibitor of soluble guanylate cyclase and nitric oxide synthase, alters lung hemodynamics and fluid filtration after endotoxin in sheep . DESIGN: Prospective, randomized, controlled experimental study with repeated measurements . SETTING: University animal laboratory . SUBJECTS: Eight yearling, awake sheep . INTERVENTIONS: Sheep were instrumented for a chronic study with vascular and lung lymph catheters . In two experiments, separated by 1 wk of recovery, the animals received intravenously either an injection of MB 10 mg/kg or a corresponding volume of 0.9% sodium chloride as pretreatment . Thirty minutes later, sheep received a bolus injection of Escherichia coli endotoxin 1 microg/kg, followed by either an infusion of MB 2.5 mg/kg/hr or a corresponding volume of 0.9% sodium chloride for 5 hrs . MEASUREMENTS AND MAIN RESULTS: MB decreased the early phase endotoxin-induced rises in pulmonary capillary pressure and pulmonary vascular resistance . MB also reduced the increments in lung lymph flow (QL) and protein clearance (CL) as well as the rightward shift of the permeability-surface area product (PS) . In addition, MB diminished the decrease in cardiac output, stabilized mean arterial pressure, and precluded the rise in plasma and lung lymph cyclic guanosine 3'-5' monophosphate . However, during the late phase, MB-treated sheep presented with a faster rise in QL with no difference in CL and PS from the endotoxemic controls . CONCLUSIONS: During the early phase of endotoxemia in sheep, MB attenuates lung injury by decreasing the enhanced lung fluid filtration as a result of reduced pulmonary capillary pressure and permeability . However, MB does not counteract the late phase increase in lung fluid filtration. Environ Mol Mutagen, 2001, 37(2), 141 - 6 Spontaneous mutation of the lacI transgene in rodents: absence of species, strain, and insertion-site influence; Zhang S et al.; Comparison of spontaneous mutation spectra derived from different transgenic constructs can provide valuable insights for interpreting the mechanisms of spontaneous mutation . In this study, spontaneous mutation frequencies and spectra of the lacI transgene are compared in the liver of C57BL/6, B6C3F1, and BC-1 mice and F344 rats . Before correction for clonal expansion, the mutant frequency varied from 2.6 +/- 0.45 to 5.0 +/- 2.4 x 10(-5) . Correction for potential clonal expansion reduced the range in mutation frequency to between 2.3 +/- 0.45 and 3.5 +/- 2.0 x 10(-5) . There is thus no statistical difference in spontaneous mutation frequency between the different strains and species . G:C --> A:T transitions and to a lesser extent, G:C --> T:A transversions dominate the mutational spectra in all of these animals . In three strains of mice, G:C --> A:T transitions account for 50.7-53.3% of mutation, 81.7-83.8% of which involve CpG sites, whereas G:C --> T:A transversions account for 17.8-32.9% of mutations with 43.2-50.0% found at CpG sites . In rats, G:C --> A:T transitions account for 38.0% of the spectra, 70.0% of which involve CpG sites, whereas G:C --> T:A transversions account for 23.0% of the spectra, 70.0% of which involve CpG sites . The distribution of other classes of mutations is also very similar . We conclude that, despite reports about species and strain differences in induced mutation, spontaneous mutations in the lacI transgene appear to be similar, regardless of genomic location, rodent strain, or species . In addition to insights into spontaneous mutation, this study also provides essential data for comparison with and interpretation of induced mutations . J Biochem Biophys Methods, 2001 Feb 26, 47(3), 233 - 7 A simple technique for the simultaneous determination of molecular weight and activity of superoxide dismutase using SDS-PAGE; Chen J et al.; A direct and rapid SDS-PAGE staining method for in situ identification of activity and molecular weight of superoxide dismutase following denaturing treatment has been developed . This technique was based on the removal of SDS after SDS-PAGE and two-step staining procedures of the SDS-polyacrylamide gel to present the achromatic activity-zones of the enzymes . We demonstrated that the detection sensitivity of SDS-PAGE staining method was the same as the traditional xanthine oxidase-NBT solution assay . Through the SDS-PAGE staining method, three classes of superoxide dismutases with distinct molecular sizes were identified in situ . Moreover, activity of copper and zinc containing superoxide dismutase in crude extracts of Escherichia coli and Actinobacillus pleuropneumoniae was significantly enhanced using the two-step staining procedure. Biochim Biophys Acta, 2001 Apr 2, 1504(2-3), 352 - 62 Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH; Roth R et al.; NADH:quinone oxidoreductase, or Complex I, is a multi-subunit membrane-bound enzyme in the respiratory chain of many pro- and eukaryotes . The enzyme catalyzes the oxidation of NADH and donates electrons to the quinone pool, coupled to proton translocation across the membrane, but the mechanism of energy transduction is not understood . In bacteria the enzyme consists of 14 subunits, seven membrane spanning and seven protruding from the membrane . The hydrophobic NuoH (NQO8, ND1, NAD1, NdhA) subunit is seemingly involved in quinone binding . A homologous, structurally and most likely functionally similar subunit is also found in F(420)H2 oxidoreductases and in complex membrane-bound hydrogenases . We have made theoretical analyses of NuoH and NuoH-like polypeptides and experimentally analyzed the transmembrane topology of the NuoH subunit from Rhodobacter capsulatus by constructing and analyzing alkaline phosphatase fusion proteins . This demonstrated that the NuoH polypeptide has eight transmembrane segments, and four highly conserved hydrophilic sequence motifs facing the inside, bacterial cytoplasm . The N-terminal and C-terminal ends are located on the outside of the membrane . A topology model of NuoH based on these results is presented, and implications from the model are discussed. Am J Respir Cell Mol Biol, 2001 Mar, 24(3), 345 - 51 Airway administration of Escherichia coli endotoxin to mice induces glucocorticosteroid-resistant bronchoconstriction and vasopermeation; Lefort J et al.; The effects of the administration of Escherichia coli endotoxin (lipopolysaccharide, LPS) into the airways of C57Bl/6 mice were studied . Neutrophil sequestration in the lungs and their enrichment, together with tumor necrosis factor (TNF)-alpha, in bronchoalveolar lavage fluid (BALF) were associated with bronchoconstriction and bronchopulmonary hyperreactivity (BHR) to methacholine and alveolocapillary dysfunction . Granulocyte depletion by the myelotoxic drug vinblastine failed to modify TNF-alpha production and prevented LPS-induced neutrophil recruitment to lungs and BALF, bronchoconstriction, and BHR . Neutrophils were again sequestered in the lungs when LPS was administered 4 to 5 d after vinblastine, whereas inhibition of their passage to BALF persisted . Under those conditions, bronchoconstriction and BHR by LPS also recovered, showing that these functional effects are independent from BALF neutrophil enrichment but require lung sequestration . Administration of granulocyte colony-stimulating factor after vinblastine counteracted its effects and allowed the recovery of lung neutrophil sequestration by LPS and a partial recovery of bronchoconstriction under conditions where neutrophils still failed to migrate to BALF . Dexamethasone (the phosphate salt and its free base) suppressed LPS-induced TNF-alpha generation in BALF and its neutrophil enrichment, whereas neutrophil lung sequestration, bronchoconstriction, BHR, and alveolocapillary dysfunction were marginally reduced and only so at low doses of dexamethasone, higher doses being inactive or aggravating . In situ neutrophil activation could account for LPS-induced bronchoconstriction and BHR, both of which are refractory to steroids and appear to be mediated by unrelated mechanisms, which may be relevant for acute respiratory distress syndrome, a condition for which LPS administration is used as a model. Development, 2001 Apr, 128(7), 1109 - 18 Establishment and maintenance of parasegmental compartments; Hughes SC et al.; Embryos of higher metazoans are divided into repeating units early in development . In Drosophila, the earliest segmental units to form are the parasegments . Parasegments are initially defined by alternating stripes of expression of the fushi-tarazu and even-skipped genes . How fushi-tarazu and even-skipped define the parasegment boundaries, and how parasegments are lost when fushi-tarazu or even-skipped fail to function correctly, have never been fully or properly explained . Here we show that parasegment widths are defined early by the relative levels of fushi-tarazu and even-skipped at stripe junctions . Changing these levels results in alternating wide and narrow parasegments . When shifted by 30% or more, the enlarged parasegments remain enlarged and the reduced parasegments are lost . Loss of the reduced parasegments occurs in three steps; delamination of cells from the epithelial layer, apoptosis of the delaminated cells and finally apoptosis of inappropriate cells remaining at the surface . The establishment and maintenance of vertebrate metameres may be governed by similar processes and properties. Cancer Res, 2001 Feb 15, 61(4), 1548 - 54 Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia; Meyer DE et al.; Elastin-like polypeptides (ELPs) are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition . They are soluble in aqueous solutions below their transition temperature (T1) but hydrophobically collapse and aggregate at temperatures greater than T1 . We hypothesized that ELPs conjugated to drugs would enable thermally targeted drug delivery to solid tumors if their T1 were between body temperature and the temperature in a locally heated region . To test this hypothesis, we synthesized a thermally responsive ELP with a T1 of 41 degrees C and a thermally unresponsive control ELP in Escherichia coli using recombinant DNA techniques . In vivo fluorescence videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors (SKOV-3 ovarian carcinoma and D-54MG glioma) implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately 2-fold increase in tumor localization compared to the same polypeptide without hyperthermia . We observed aggregates of the thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in control experiments, which demonstrates that the phase transition of the thermally responsive ELP carrier can be engineered to occur in vivo at a specified temperature . By exploiting the phase transition-induced aggregation of these polypeptides, this method provides a new way to thermally target polymer-drug conjugates to solid tumors. Cancer Res, 2001 Feb 15, 61(4), 1338 - 46 Down-regulation of promoter 1.3 activity of the human aromatase gene in breast tissue by zinc-finger protein, snail (SnaH); Okubo T et al.; Aromatase (estrogen synthetase) is expressed in breast cancer tissue, and in situ expression of the enzyme stimulates breast cancer growth . Promoter I.3 is one of the major promoters that control the expression of aromatase in breast cancer tissue . Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that the zinc-finger transcriptional factor Snail (SnaH) interacted with a regulatory region near promoter I.3 of the human aromatase gene . DNA mobility shift assays and mutation analyses using recombinant SnaH protein expressed in Escherichia coli have revealed that this protein interacts with a segment, 5'-CTGATGAAGT-3', which is between 66 and 76 bp upstream from the transcriptional start site of promoter I.3 . Using mammalian cell transfection experiments, SnaH was found to act as a repressor of promoter I.3 activity . Site-directed mutagenesis experiments have revealed that the NH2-terminal SNAG domain is important for the repressor activity of SnaH . To demonstrate the inhibitory activity against aromatase expression, a stable SnaH-expressing MDA-MB-231 breast cancer cell line was generated, and the aromatase RNA messages in the SnaH-transfected cell line were found to be 30% of those in the vector-transfected cell line . Reverse transcription-PCR analysis on RNAs isolated from 12 cell lines has confirmed that SnaH is expressed at a higher level in normal breast epithelial cell and stromal fibroblast cell lines than in breast cancer cell lines . In addition, SnaH mRNA was detected in only 16 of 55 breast cancer specimens . On the other hand, aromatase mRNA was detected in 54 of the 55 specimens . Our results indicate that SnaH acts as a repressor that down-regulates the expression of aromatase in normal breast tissue by suppressing the function of promoter I.3 . A reduction of the expression of SnaH in breast cancer tissue further suggests a cancer-protective role for this protein in normal breast tissue. J Pept Sci, 2001 Jan, 7(1), 50 - 7 Expression, refolding and indirect immobilization of horseradish peroxidase (HRP) to cellulose via a phage-selected peptide and cellulose-binding domain (CBD); Levy I et al.; We examined the potential immobilization of horseradish peroxidase (HRP) to cellulose with cellulose-binding domain (CBD) as a mediator, using a ligand selected from a phage-displayed random peptide library . A 15-mer random peptide library was panned on cellulose-coated plates covered with CBD in order to find a peptide that binds to CBD in its bound form . The sequence I/LHS, which was found to be an efficient binder of CBD, was fused to a synthetic gene of HRP as an affinity tag . The tagged enzyme (tHRP) was then immobilized on microcrystalline cellulose coated with CBD, thereby demonstrating the indirect immobilization of a protein to cellulose via three amino acids selected by phage display library and CBD. Zhonghua Bing Li Xue Za Zhi, 1998 Dec, 27(6), 412 - 5 {The killing effects of two prodrug sensitivity genes on human pancreatic carcinoma cells PC-2}; Zhang L et al.; OBJECTIVE: To compare the killing effects of herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system versus cytosine deaminase (CD)/5-fluorocytosine (5-FC) system on human pancreatic carcinoma PC-2 cells . METHODS: Recombinant retroviral vectors expressing HSV-TK and CD genes were constructed and transduced into pancreatic carcinoma cell line . Prodrug sensitivity and IC50 values (concentration of drug at which cell growth is inhibited by 50%) of the transduced cells were measured by MTT method . The bystander effects in the two systems were also compared . RESULTS: The IC50 value of HSV-TK-transduced cells to GCV was (1.06 +/- 0.12) micromol/L and 558-fold lower than parental PC-2 cells, while the IC50 value of CD-transduced cells to 5-FC.00 was (33.00 + 0.95) micromol/L and 258-fold lower than parental PC-2 cells . Mixed cells containing 10% of transduced cells showed 39% and 50.3% growth inhibition in TK/GCV and CD/5-FC systems respectively . CONCLUSION: Both HSV-TK/GCV and CD/5-FC systems showed effective antitumor activity in vitro to pancreatic carcinoma PC-2 cells . The therapeutic index of HSV-TK/GCV system is higher, but its bystander effect is lower than that of CD/5-FC system. J Mol Evol, 2001 Feb, 52(2), 205 - 14 Aquifex aeolicus 3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthase: a new class of KDO 8-P synthase? Birck MR, Woodard RW. The relationship between 3-deoxy-D-manno-2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature . Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes . Using a maximum likelihood-based grouping of 29 KDO 8-P synthase sequences, we demonstrate the existence of a new class of KDO 8-P synthase, the members of which we propose to require a metal cofactor for catalysis . Similarly, we hypothesize a class of DAH 7-P synthase that does not have the metal requirement of the heretofore model E . coli enzyme . Based on this information and a careful investigation of the reported X-ray crystal structures, we also propose that KDO 8-P synthase and DAH 7-P synthase are the product of a divergent evolutionary process from a common ancestor. Plant Physiol, 2001 Mar, 125(3), 1508 - 16 Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice; Sakamoto T et al.; A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase . We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L . cv Nipponbare) that encodes a GA 2-oxidase . The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively . Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs . These transgenic plants were deficient in endogenous GA(1) . These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo . OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems . In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves . This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth . It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase . These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem. J Bacteriol, 2001 Apr, 183(7), 2335 - 42 Engineered fatty acid biosynthesis in Streptomyces by altered catalytic function of beta-ketoacyl-acyl carrier protein synthase III; Smirnova N et al.; The Streptomyces glaucescens beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers . This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process . Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis . Plasmid-based expression of these mutants in S . glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain . In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles . These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor) . Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D(3) acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII . These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant . To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed . In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro . The Cys122Ala mutant exhibited higher activity . This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 microM), a branched-chain fatty acid biosynthetic precursor . Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase . These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S . glaucescens. J Bacteriol, 2001 Apr, 183(7), 2280 - 8 Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii; Simao RC et al.; The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized . The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM . B . emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease . In the presence of Ca(2+), B . emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain . CaM expression is developmentally regulated in B . emersonii, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium . Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination . The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B . emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively . All these drugs also inhibited growth and zoospore production in this fungus . The Ca(2+) channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth . The data presented suggest that the Ca(2+)-CaM complex and CaMKII play an important role during growth and sporulation in B . emersonii. J Bacteriol, 2001 Apr, 183(7), 2273 - 9 Glucan synthase complex of Aspergillus fumigatus; Beauvais A et al.; The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated . The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A . fumigatus were cloned and sequenced . Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans . Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli . Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans . AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus . AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p . Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters. J Bacteriol, 2001 Apr, 183(7), 2265 - 72 Global impact of sdiA amplification revealed by comprehensive gene expression profiling of Escherichia coli; Wei Y et al.; In Escherichia coli the amplification of sdiA, a positive activator of ftsQAZ, genes that are essential for septation, results in mitomycin C resistance . To help us understand this resistance phenotype, genes whose expression was altered by increased sdiA dosage were identified using a DNA microarray-based, comprehensive transcript profiling method . The expression of 62 genes was reduced by more than threefold; of these, 41 are involved in motility and chemotaxis . Moreover, the expression of 75 genes, 36 of which had been previously characterized, was elevated at least threefold . As expected, increased sdiA dosage led to significantly elevated sdiA and 'ddlB-ftsQAZ-lpxC operon expression . Transcription of two genes, uvrY and uvrC, located downstream of sdiA and oriented in the same direction, was elevated about 10-fold, although the intervening gene, yecF, of opposite polarity was unaffected by increased sdiA dosage . Three genes (mioC and gidAB) flanking the replication origin, oriC, were transcribed more often when sdiA dosage was high, as were 12 genes within 1 min of a terminus of replication, terB . Transcription of the acrABDEF genes, mapping in three widely spaced loci, was elevated significantly, while several genes involved in DNA repair and replication (e.g., nei, recN, mioC, and mcrC) were moderately elevated in expression . Such global analysis provides a link between septation and the response to DNA-damaging agents. J Bacteriol, 2001 Apr, 183(7), 2259 - 64 In vivo titration of mitomycin C action by four Escherichia coli genomic regions on multicopy plasmids; Wei Y et al.; Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli . MMC resistance is caused by the presence of any of four distinct E . coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors . mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance . The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps . SdiA is a transcriptional activator of ftsQAZ genes involved in cell division. J Bacteriol, 2001 Apr, 183(7), 2219 - 25 Determination of Wolbachia genome size by pulsed-field gel electrophoresis; Sun LV et al.; Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases . Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb) . The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria . There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes . The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined . They also indicated that the Wolbachia genome is circular. J Bacteriol, 2001 Apr, 183(7), 2204 - 11 Genetic evidence that the alpha5 helix of the receiver domain of PhoB is involved in interdomain interactions; Allen MP et al.; Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals . Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein . Generally, response regulators are made up of a receiver domain and an output domain . Phosphorylation of the receiver domain modulates the activity of the output domain . The mechanisms by which receiver domains control the activities of their respective output domains are unknown . To address this question for the PhoB protein from Escherichia coli, we have employed two separate genetic approaches, deletion analysis and domain swapping . In-frame deletions were generated within the phoB gene, and the phenotypes of the mutants were analyzed . The output domain, by itself, retained significant ability to activate transcription of the phoA gene . However, another deletion mutant that contained the C-terminal alpha-helix of the receiver domain (alpha5) in addition to the entire output domain was unable to activate transcription of phoA . This result suggests that the alpha5 helix of the receiver domain interacts with and inhibits the output domain . We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB . A chimera that joins the N-terminal approximately 85% of CheY's receiver domain to the beta5-alpha5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity . The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the alpha5 helix of its unphosphorylated receiver domain and its output domain. J Bacteriol, 2001 Apr, 183(7), 2165 - 71 Viability of rep recA mutants depends on their capacity to cope with spontaneous oxidative damage and on the DnaK chaperone protein; Bredeche MF et al.; Replication arrests due to the lack or the inhibition of replicative helicases are processed by recombination proteins . Consequently, cells deficient in the Rep helicase, in which replication pauses are frequent, require the RecBCD recombination complex for growth . rep recA mutants are viable and display no growth defect at 37 or 42 degrees C . The putative role of chaperone proteins in rep and rep recA mutants was investigated by testing the effects of dnaK mutations . dnaK756 and dnaK306 mutations, which allow growth of otherwise wild-type Escherichia coli cells at 40 degrees C, are lethal in rep recA mutants at this temperature . Furthermore, they affect the growth of rep mutants, and to a lesser extent, that of recA mutants . We conclude that both rep and recA mutants require DnaK for optimal growth, leading to low viability of the triple (rep recA dnaK) mutant . rep recA mutant cells form colonies at low efficiency when grown to exponential phase at 30 degrees C . Although the plating defect is not observed at a high temperature, it is not suppressed by overexpression of heat shock proteins at 30 degrees C . The plating defect of rep recA mutant cells is suppressed by the presence of catalase in the plates . The cryosensitivity of rep recA mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures. J Struct Biol, 2000 Dec, 132(3), 241 - 50 Structural analysis of F18 fimbriae expressed by porcine toxigenic Escherichia coli; Hahn E et al.; The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes . The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA . The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm . Analyzing their helical symmetry showed the axially repeating units to alternate in a "zigzag" manner around the helical axis with an axial rise of 2.2 nm . Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed . Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae . Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF . The presence of the latter two proteins might cause the observed 27-nm axial pattern . Biochem Biophys Res Commun, 2001 Mar, 281(5), 1321 - 4 The dipeptide, gamma-glutamylcysteine, is recognized by the anti-glutathione antibody single chain Fv fragment 20C9; Horibe T et al.; The anti-glutathione antibody scFv 20C9, which we previously isolated from a human synthetic phage antibody scFv library {Hirose, M., Hayano, T., Shirai, H., Nakamura, H., and Kikuchi, M . (1998) Protein Eng . 11, 243-248}, was expressed in the E . coli pET system and purified by sequential chromatography on Ni and glutathione-conjugated affinity resins . The purified scFv 20C9 antibody was characterized for its binding affinity for several glutathione derivatives by the BIACORE system . Although GSH, GSSG, and gamma-Glu-Cys could bind to the immobilized antibody, this was not the case for Cys-Gly, l-Glu, l-Cys, l-Gly, or several other glutathione derivatives such as gamma-Glu-Ser-Gly . The results suggest that a gamma-glutamic acid and sulfur atom are important for scFv 20C9 antibody recognition of glutathione . This is the first report to indicate that an scFv antibody can recognize a region as small as a dipeptide . Biochem Biophys Res Commun, 2001 Mar, 281(5), 1271 - 6 Alteration of inhibitory properties of Pleurotus ostreatus proteinase A inhibitor 1 by mutation of its C-terminal region; Kojima S et al.; Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins . In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene . The wild-type POIA1 was found to inhibit subtilisin BPN' with an inhibitor constant (K(i)) of 3.2 x 10(-9) M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN' was used as a target protease . Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN' . The mutated POIA1 inhibited subtilisin BPN' with a K(i) value of 2.8 x 10(-11) M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease . These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation . Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region . Biochem Biophys Res Commun, 2001 Mar, 281(5), 1256 - 60 A single nucleotide polymorphism of CYP2b6 found in Japanese enhances catalytic activity by autoactivation; Ariyoshi N et al.; A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese . The frequency of the variant allele was found to be 19.9% . The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity . The wild-type enzyme showed typical Michaelis-Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher V(max) value compared to that of the wild-type enzyme . Eadie-Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant . This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity . Methods, 2001 Mar, 23(3), 218 - 32 Phenanthroline-Cu(II) cleavage as a probe of rRNA structure; Muth GW et al.; Chemical cleavage is developing into a powerful tool for analysis and characterization of nucleic acids . Phenanthroline-Cu(II) cleavage has been used extensively for studies of DNA for the last two decades, but recently has been applied to structural studies of RNA as well . This approach has been used to study the structure and structural changes occurring in ribosomal RNA within the ribosomes . In this article we discuss the mechanism by which phenanthroline cleaves, the applications possible using this approach, and the results that can be obtained . Protocols for use of phenanthroline are outlined as well. J Mol Biol, 2001 Mar 16, 307(1), 465 - 77 Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis; Mendel S et al.; Valine inhibition of acetohydroxyacid synthase (AHAS) plays an important role in regulation of biosynthesis of branched-chain amino acids in bacteria . Bacterial AHASs are composed of separate catalytic and regulatory subunits; while the catalytic subunits appear to be homologous with several other thiamin diphosphate-dependent enzymes, there has been no model for the structure of the small, regulatory subunits (SSUs) . AHAS III is one of three isozymes in Escherichia coli . Its large subunit (encoded by ilvI) by itself has 3-5 % activity of the holoenzyme and is not sensitive to inhibition by valine . The SSU (encoded by ilvH) associates with the large subunit and is required for full catalytic activity and valine sensitivity . The isolated SSU binds valine . The properties of several mutant SSUs shed light on the relation between their structure and regulatory function . Three mutant SSUs were obtained from spontaneous Val(R) bacterial mutants and three more were designed on the basis of an alignment of SSU sequences from valine-sensitive and resistant isozymes, or consideration of the molecular model developed here . Mutant SSUs N11A, G14D, N29H and A36V, when reconstituted with wild-type large subunit, lead to a holoenzyme with drastically reduced valine sensitivity, but with a specific activity similar to that of the wild-type . The isolated G14D and N29H subunits do not bind valine . Mutant Q59L leads to a valine-sensitive holoenzyme and isolated Q59L binds valine . T34I has an intermediate valine sensitivity . The effects of mutations on the affinity of the large subunits for SSUs also vary . D . Fischer's hybrid fold prediction method suggested a fold similarity between the N terminus of the ilvH product and the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase . On the basis of this prediction, together with the properties of the mutants, a model for the structure of the AHAS SSUs and the location of the valine-binding sites can be proposed . J Mol Biol, 2001 Mar 16, 307(1), 379 - 91 Detection of two partially structured species in the folding process of the amyloidogenic protein beta 2-microglobulin; Chiti F et al.; beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis . The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops . These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays . All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms) . The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively . Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds . Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules . Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state . The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus . Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation . J Mol Biol, 2001 Mar 16, 307(1), 341 - 56 Refined structures of beta-ketoacyl-acyl carrier protein synthase III; Qiu X et al.; beta-Ketoacyl-acyl carrier protein synthase III (FabH) is a condensing enzyme that plays central roles in fatty acid biosynthesis . Three-dimensional structures of E . coli FabH in the presence and absence of ligands have been refined to 1.46 A resolution . The structures of improved accuracy revealed detailed interactions involved in ligand binding . These structures also provided new insights into the FabH mechanism, e.g . the possible role of a water or hydroxyl anion in Cys112 deprotonation . A structure of the apo enzyme uncovered large conformational changes in the active site, exemplified by the disordering of four essential loops (84-86, 146-152, 185-217 and 305-307) and the movement of catalytic residues (Cys112 and His244) . The disordering of the loops leads to greater than 50 % reduction in the FabH dimer interface, suggesting a dynamic nature for an unusually large portion of the dimer interface . The existence of a large solvent-accessible channel in the dimer interface as well as two cis-peptides (cis-Pro88 and cis-Phe308) in two of the disordered loops may explain the observed structural instabilities . J Mol Biol, 2001 Mar 16, 307(1), 229 - 46 Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle; Batey RT et al.; The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya . The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein . Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition . To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants . This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the protein . J Mol Biol, 2001 Mar 16, 307(1), 213 - 28 Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA . III . Functional analysis of the 690 loop; Morosyuk SV et al.; An instant-evolution experiment was performed on the eight nucleotides comprising the loop region of the 690 hairpin in Escherichia coli 16 S ribosomal RNA . Positions 690 to 697 were randomly mutated and 101 unique functional mutants were isolated, sequenced and analyzed for function in vivo . Non-random nucleotide distributions were observed at each of the mutated positions except 693 and 694 . Nucleotide identity significantly affected ribosome function at positions 690, 695, 696 and 697 . Pyrimidines were absent at position 696 in the instant-evolution pool as were C at position 691 and G at position 697 . A highly significant covariation was observed between nucleotides 690 and 697 . No functional double mutants at positions 691 and 696 were obtained from the instant-evolution pool . In our NMR structure of the 690 loop, both the G690.U697 and G691.A696 form sheared hydrogen-bonded mismatches . To further examine the functional constraints between these paired nucleotides, one set of site-directed mutations was constructed at positions 690:697 and another set was constructed at positions 691:696 . Functional analysis of the site-directed mutants is consistent with our instant-evolution findings and revealed constraints on the placement of specific functional groups observed in the NMR structure . Ten instant-evolution mutants were isolated that are more functional than the wild-type . Hyperactivity in these mutants correlates with a single mutation at position 693 . J Mol Biol, 2001 Mar 16, 307(1), 197 - 211 Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA . II . NMR solution structure; Morosyuk SV et al.; The solution structure of the conserved 690 hairpin from Escherichia coli 16 S rRNA was determined by NMR spectroscopy . The 690 loop is located at the surface of the 30 S subunit in the platform region and has been implicated in interactions with P-site bound tRNA, E-site mRNA, S11 binding, IF3 binding, and in RNA-RNA interactions with the 790 loop of 16 S rRNA and domain IV of 23 S rRNA . The structure reveals a novel sheared type G690.U697 base-pair with a single hydrogen bond from the G690 amino to U697-04 . G691 and A696 also form a sheared pair and U692 forms a U-turn with an H-bond to the A695 non-bridging phosphate oxygen . The sheared pairs and U-turn result in the continuous single-stranded stacking of five residues from 6693 to U697 with their Watson-Crick functional groups exposed in the minor groove . The overall fold of the 690 hairpin is similar to the anticodon loop of tRNA . The structure provides an explanation for chemical protection patterns in the loop upon interaction with tRNA, the 50 S subunit, and S11 . In vivo genetic studies demonstrate the functional importance of the motifs observed in the solution structure of the 690 hairpin . J Mol Biol, 2001 Mar 16, 307(1), 173 - 82 Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1; Chen XS et al.; The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly . Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein . Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis . For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation . The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested . The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design . J Mol Biol, 2001 Mar 16, 307(1), 119 - 35 Determinants of chemotactic signal amplification in Escherichia coli; Kim C et al.; A well-characterized protein phosphorelay mediates Escherichia coli chemotaxis towards the amino acid attractant aspartate . The protein CheY shuttles between flagellar motors and methyl-accepting chemoreceptor (MCP) complexes containing the linker CheW and the kinase CheA . CheA-CheY phosphotransfer generates phospho-CheY, CheY-P . Aspartate triggers smooth swim responses by inactivation of the CheA bound to the target MCP, Tar; but this mechanism alone cannot explain the observed response sensitivity . Here, we used behavioral analysis of mutants deleted for CheZ, a catalyst of CheY-P dephosphorylation, or the methyltransferase CheR and/or the methylesterase CheB to examine the roles of accelerated CheY-P dephosphorylation and MCP methylation in enhancement of the chemotactic response . The extreme motile bias of the mutants was adjusted towards wild-type values, while preserving much of the aspartate response sensitivity by expressing fragments of the MCP, Tsr, that either activate or inhibit CheA . We then measured responses to small jumps of aspartate, generated by flash photolysis of photo-labile precursors . The stimulus-response relation for Delta cheZ mutants overlapped that for the host strains . Delta cheZ excitation response times increased with stimulus size consistent with formation of an occluded CheA state . Thus, neither CheZ-dependent or independent increases in CheY-P dephosphorylation contribute to the excitation response . In Delta cheB Delta cheR or Delta cheR mutants, the dose for a half-maximal response, {Asp}(50), was ca 10 microM; but was elevated to 100 microM in Delta cheB mutants . In addition, the stimulus-response relation for these mutants was linear, consistent with stoichiometric inactivation, in contrast to the non-linear relation for wild-type E . coli . These data suggest that response sensitivity is controlled by differential binding of CheR and/or CheB to distinct MCP signaling conformations . J Mol Biol, 2001 Mar 16, 307(1), 93 - 105 The hydH/G Genes from Escherichia coli code for a zinc and lead responsive two-component regulatory system; Leonhartsberger S et al.; The hydH/G genes from Escherichia coli code for a two-component regulatory system that has been implicated in the regulation of hydrogenase 3 formation . In a detailed study of the function of HydH/G employing hycA'-'lacZ reporter gene fusions, it was shown that HydH/G indeed led to a stimulation of activation of the hycA promoter responsible for hydrogenase 3 synthesis but only when hydG is overexpressed from a plasmid in a strain lacking FhlA . Since the stimulation was not observed with an fdhF'-'lacZ fusion, and since it was independent from a functional hydH gene product, it must be considered as unspecific cross-talk . An extensive search for the actual physiological signal of HydH/G showed that the system responds to high concentrations of zinc or lead in the medium . Expression of zraP, a gene inversely oriented to hydH/G whose product seems to be involved in acquisition of tolerance to high Zn(2+) concentrations, is stimulated by high Zn(2+) and Pb(2+) concentrations and this stimulation requires both HydH and HydG . Purified HydG in the presence of phosphoryl donors binds to a region within the zraP-hydHG intergenic region that is characterised by two inverted repeats separated by a 14 bp spacer . Putative -12/-24 sigma(54)-dependent promoter motifs are present upstream of both the zraP and the hydHG transcriptional units; in accordance, transcription of zraP is strictly dependent on the presence of a functional rpoN gene . The expression of hydH/G is autoregulated: high Zn(2+) and Pb(2+) concentrations lead to a significant increase of the HydG protein content which took place only in a hydH(+) genetic background . Since HydH binds to membranes tightly, it is assumed that the HydH/G system senses high periplasmic Zn(2+) and Pb(2+) concentrations and contributes to metal tolerance by activating the expression of zraP . The redesignation of hydH/G as zraS/R is suggested . J Mol Biol, 2001 Mar 16, 307(1), 39 - 49 Modulation of transcription reveals a new mechanism of triplet repeat instability in Escherichia coli; Schumacher S et al.; Many human hereditary disease genes are associated with the expansion of triplet repeat sequences . In Escherichia coli (CTG/CAG) triplet repeat sequences are unstable and we have developed a plasmid-based assay enabling us to observe and quantify both expansions and deletions . In this work, we have investigated the role of transcription on the instability of a (CTG/CAG) insert containing 64 repeats . Using this assay, we show that induction of transcription results in a significant increase in the frequency of long deletions and a reduction in the frequency of long expansions . On the other hand, overproduction of transcription repressor molecules leads to an increase in both expansions and deletions . In this latter case, we propose that the increased instability is due to the arrest of replication progression by the interaction of the repressor molecule with its cognate operator and subsequent generations of DNA strand breaks . J Mol Biol, 2001 Mar 16, 307(1), 25 - 30 Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in initiating transcription complexes; Brodolin K et al.; Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)) . RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)) . We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift . The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale . However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes . The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site . J Mol Biol, 2001 Mar 16, 307(1), 1 - 8 Crystal structure of the catalytic core component of the alkylhydroperoxide reductase AhpF from Escherichia coli; Bieger B et al.; Alkylhydroperoxide reductases (AhpR, EC 1.6.4.*) are essential for the oxygen tolerance of aerobic organisms by converting otherwise toxic hydroperoxides of lipids or nucleic acids to the corresponding alcohols . The AhpF component belongs to the family of pyridine nucleotide-disulphide oxidoreductases and channels electrons from NAD(P)H towards the AhpC component which finally reduces cognate substrates . The structure of the catalytic core of the Escherichia coli AhpF (A212-A521) with a bound FAD cofactor was determined at 1.9 A resolution in its oxidized state . The dimeric arrangement of the AhpF catalytic core and the predicted interaction mode between the N-terminal PDO-like domain and the NADPH domain favours an intramolecular electron transfer between the two redox-active disulphide centres of AhpF . J Mol Biol, 2001 Mar 2, 306(4), 863 - 76 How methionyl-tRNA synthetase creates its amino acid recognition pocket upon L-methionine binding; Serre L et al.; Amino acid selection by aminoacyl-tRNA synthetases requires efficient mechanisms to avoid incorrect charging of the cognate tRNAs . A proofreading mechanism prevents Escherichia coli methionyl-tRNA synthetase (EcMet-RS) from activating in vivo L-homocysteine, a natural competitor of L-methionine recognised by the enzyme . The crystal structure of the complex between EcMet-RS and L-methionine solved at 1.8 A resolution exhibits some conspicuous differences with the recently published free enzyme structure . Thus, the methionine delta-sulphur atom replaces a water molecule H-bonded to Leu13N and Tyr260O(eta) in the free enzyme . Rearrangements of aromatic residues enable the protein to form a hydrophobic pocket around the ligand side-chain . The subsequent formation of an extended water molecule network contributes to relative displacements, up to 3 A, of several domains of the protein . The structure of this complex supports a plausible mechanism for the selection of L-methionine versus L-homocysteine and suggests the possibility of information transfer between the different functional domains of the enzyme . J Mol Biol, 2001 Mar 2, 306(4), 851 - 61 Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site; Sam MD et al.; Crystal structures of EcoRV endonuclease bound in a ternary complex with cognate duplex DNA and manganese ions have previously revealed an Mn(2+)-binding site located between the enzyme and the DNA outside of the dyad-symmetric GATATC recognition sequence . In each of the two enzyme subunits, this metal ion bridges between a distal phosphate group of the DNA and the imidazole ring of His71 . The new metal- binding site is specific to Mn(2+) and is not occupied in ternary cocrystal structures with either Mg(2+) or Ca(2+) . Characterization of the H71A and H71Q mutants of EcoRV now demonstrates that these distal Mn(2+) sites significantly modulate activity toward both cognate and non-cognate DNA substrates . Single-turnover and steady-state kinetic analyses show that removal of the distal site in the mutant enzymes increases Mn(2+)-dependent cleavage rates of specific substrates by tenfold . Conversely, the enhancement of non-cognate cleavage at GTTATC sequences by Mn(2+) is significantly attenuated in the mutants . As a consequence, under Mn(2+) conditions EcoRV-H71A and EcoRV-H71Q are 100 to 700-fold more specific than the wild-type enzyme for cognate DNA relative to the GTTATC non-cognate site . These data reveal a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 A distant from the scissile phosphodiester linkages . They also show that discrimination of cognate versus non-cognate DNA sequences by EcoRV depends in part on contacts with the sugar-phosphate backbone outside of the target site . J Mol Biol, 2001 Mar 2, 306(4), 669 - 79 Functional cooperation between topoisomerase I and single strand DNA-binding protein; Sikder D et al.; Protein-protein interactions play important role in cell biochemistry by favorably or adversely influencing major molecular events . In most documented cases, the interaction is direct between the partner molecules . Influence of activity in the absence of direct physical interaction between DNA transaction proteins is another important means of modulation . We show here that single strand binding protein stimulates DNA topoisomerase I activity without direct protein-protein interactions . The stimulation is specific to topoisomerase I, as DNA gyrase activity is unaffected by SSB . We propose that such cases of functional collaboration between DNA transaction proteins play important roles in vivo . Mol Genet Metab, 2001 Mar, 72(3), 269 - 72 Congenital lactic acidosis: evaluation of the properties of the a199t natural variant of human pyruvate dehydrogenase e1alpha by in vitro mutation; Wu YG et al.; One cause of congenital lactic acidosis is a mutation in the E1 alpha-subunit of the pyruvate dehydrogenase multienzyme complex . Little is known about the consequences of these mutations at the enzymatic level . Here we study the A199T mutation by expressing the protein in Escherichia coli . The specific activity is 25% of normal and the K(m) for pyruvate is elevated by 10-fold . Inhibitors of lactate dehydrogenase might be a useful therapy for patients with such mutations . J Vet Diagn Invest, 2001 Jan, 13(1), 22 - 5 Polyclonal sandwich ELISAs to detect F18 fimbriated Escherichia coli in pigs in Northern Ireland; Bell C et al.; F18 fimbriated Escherichia coli are a newly described cause of postweaning diarrhea in pigs . Polyclonal rabbit antisera were raised to the antigenic variants, F18ab and F18ac, of these fimbriae and were used to develop monospecific sandwich enzyme-linked immunosorbent assays (ELISAs) . The ELISAs were standardized with type cultures characterized by polymerase chain reaction techniques (PCR) and then used to conduct a study of the prevalence of F18 fimbriated E . coli in pigs in Northern Ireland . A total of 176 isolates were tested by ELISA and PCR . Eight isolates were positive for F18 by ELISA, of which 2 were shown to be false positives by PCR and one was PCR positive but ELISA negative . Of the 6 confirmed ELISA positives, all produced VT2 toxin and 3 produced ST toxin . Four positives were from serogroups O138 and O139, previously associated with porcine diarrhea. Zhonghua Xue Ye Xue Za Zhi, 1998 Jun, 19(6), 294 - 8 {Therapeutic effects of combined suicide gene and cytokine gene therapy on erythroleukemia-bearing mice}; Ju D et al.; OBJECTIVE: To explore the antitumor effect of combined transfer of suicide gene and GM-CSF gene in erythroleukemia-bearing mice . METHODS: Adenovirus harboring E . coli . cytosine deaminase (CD) gene (Ad-CD) and/or GM-CSF gene (Ad-GM-CSF) were used for the treatment of erythroleukemia-bearing mice . The mice were inoculated with FBL-3 erythroleukemia cells subcutaneously and 3 days later received Ad-CD followed by 5-fluorocytosine (5FC) treatment with or without Ad-GM-CSF . RESULTS: The mice received Ad-CD/5FC and Ad-GM-CSF developed tumors more slowly and survived much longer than those received Ad-CD/5FC alone, Ad-GM-CSF alone, control virus Ad-LacZ/5FC or PBS . Combined transfer of CD gene and GM-CSF gene induced a higher specific CTL activity than control therapies did . Pathological examination illustrated that there were tumor necrosis and massive lymphocyte infiltration in the mice after the combined therapy . CONCLUSION: Combined transfer of suicide gene and cytokine gene could synergistically inhibit the growth of erythroleukemia cells in the mice and induce tumor specific immunity of the host. Chin Med Sci J, 1997 Mar, 12(1), 15 - 21 PCR amplification, molecular cloning, DNA sequence analysis and immuno/protection in BALB/C mice of the 33 kDa endoflagellar protein of L . interrorgans serovar lai; Dai B et al.; A pair of oligonucleotide primers were designed to amplify the endoflagella gene of L . interrogans serovar lai . An approximately 840 bp fragment was generated with PCR and inserted into plasmid pUC8, after the fragment and pUC8 were digested respectively with BamHI and PstI . A recombinant plasmid (designated as pLF1) was obtained . SDS-PAGE analysis indicated that a 33 kDa was expressed in E . coli JM103 harboring pLF1 and the expression level of the protein was 11% of the total bacterial soluble proteins . Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflegella (Axial filament) of Leptospira interrogans serover lai . Nucleotide sequence data showed an open reading frame encoding 282 aminoacids residues, corresponding to a protein of molecular weight 33.6 kDa . Comparison of the deduced endoflagellar subunit protein (flaB) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum flaB proteins . Immunization/protection experiment was performed on the model of BALB/C mice and showed that there was higher survival rate in the group JM103-pLE1 than in the group JM103-pUC8. Nature, 2001 Mar 1, 410(6824), 112 - 6 A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis; Srinivasula SM et al.; X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP . Here we show that XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315 . Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevented caspase-9 association with XIAP and its concomitant inhibition . We note that the N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs . Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3 . Our studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis. Eur J Immunol, 2001 Mar, 31(3), 716 - 25 Profiling the immune response in patients with breast cancer by phage-displayed cDNA libraries; Sioud M et al.; Display on the surface of filamentous phages has been shown to be well suited for the enrichment of serum antibody-binding ligands . Here, we have taken the advantage of this technology to analyze the humoral immune response in patients with cancer . The cDNA repertoires from breast cancer cell lines T47D and MCF-7 were fused to the 3'-end of the filamentous phage M13 gene VI in all three reading frames . When the libraries were biopanned on rabbit polyclonal IgG against the human Bcl-x(L) protein, positive clones were selected, thus confirming the utility of the libraries . Using serum antibodies from patients with breast cancer, we specifically selected IgG-binding phage-encoded cDNA products . Sequence analysis of the selected clones identified important antigens including p53, centromere-F, int-2, pentraxin I, integrin beta5, cathepsin L2 and S3 ribosomal protein . The selected phage-displayed cDNA products were recognized by a significant number of breast cancer sera as compared to sera from normal individuals . Although the human pentraxin I mRNA was reported to be exclusively localized in the nervous system, we found it also expressed by breast cancer cell lines . Four out of 30 patients with breast cancer (13 %) showed reactivity with the recombinant pentraxin expressed in Escherichia coli, while no reactivity was found in normal sera . The obtained results demonstrate that phage display could be a valuable method for the identification of antigens recognized by the humoral immune system in patients with cancer. Biopolymers, 2000, 55(5), 399 - 406 Identification of the ligand-binding site of the BMP type IA receptor for BMP-4; Hatta T et al.; Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines . BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors . To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis . The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase . Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures . The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor . All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) >> T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold) . The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants . These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule . The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet . These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily. Parasite Immunol, 2001 Mar, 23(3), 111 - 9 Oestrus ovis (Diptera: Oestridae): effects of larval excretory/secretory products on nitric oxide production by murine RAW 264.7 macrophages; Tabouret G et al.; Larvae of Oestrus ovis (Insecta: Diptera: Oestridae) are common parasites of nasal and sinus cavities of sheep and goats . Previous studies revealed that crude extracts of larvae modify NO synthesis by ovine monocyte derived macrophages . The aim of this study was to investigate the larval excretory/secretory products effects on nitric oxide production by murine tumour macrophages RAW 264.7 . Stimulation of RAW macrophages by excretory/secretory products of the three instars larvae (25 microg/ml) significantly increased nitrite concentrations in culture supernatants compared to negative and positive Escherichia coli lipopolysaccharide control . This effect was time and dose dependent . Nitrite production in culture supernatants was due to induction of isoform NOS-2 because both NG monomethyl L-arginine (100 microM) and dexamethasone (20 microM) inhibited, by 60 and 50%, respectively, nitrite accumulation in culture supernatants . First steps of purification, by ion exchange chromatography, indicated that one protein of 29 kDa was able to induce NO synthesis by macrophages . Further studies are needed for a better characterization of these molecule and to investigate their immunogenicity for a vaccine approach. Int J Biochem Cell Biol, 2001 Feb, 33(2), 155 - 62 The optical interconversion of the P-450 and P-420 forms of neuronal nitric oxide synthase: effects of sodium cholate, mercury chloride and urea; Jiang H et al.; We investigated whether or not neuronal nitric oxide synthase (nNOS) (EC 1.14.13.39) was converted to the P-420 form on exposure to sodium cholate, mercury chloride or urea, and the reconversion of the P-420 to the P-450 form . Sodium cholate and mercury chloride induced the conversion of nNOS from the P-450 to the P-420 form in concentration- and incubation time-dependent manners, and the nNOS activity decreased . In the presence of glycerol, L-arginine and/or tetrahydrobiopterin, the sodium cholate-treated P-420 form could be reconverted to the P-450 form under constant experimental conditions, and the nNOS activity could also be restored . The mercury chloride-treated P-420 form of nNOS could be reconverted to the P-450 form on incubation with reduced glutathione (GSH) or L-cysteine, and the nNOS activity was recovered . However, no reconversion of the mercury chloride-treated P-420 form to the P-450 form was observed in the presence of glycerol, L-arginine, or tetrahydrobiopterin . Urea (4.0 M) dissociated nNOS into its subunits, but nNOS remained in the P-450 form . The nNOS monomer was more susceptible to sodium cholate . After removing the urea by dialysis, and supplementation of the nNOS solution with glycerol, L-arginine or BH(4), the P-420 was reconverted to the P-450 form, and the reassociation of nNOS monomers was also observed . These results suggested that nNOS was more stable as to exposure to sodium cholate, mercury chloride or urea in comparison to microsomal cytochrome P-450, which may be due to the different heme environment and protein structure. J Therm Biol, 2001 Jun, 26(3), 159 - 163 Miniature data loggers for remote measurement of body temperature in medium-sized rodents; Kamerman PR et al.; We have investigated the use of miniature temperature data loggers for the recording of abdominal temperature in laboratory animals, using the guinea pig as a model . The data loggers, which are small (16cm(3)), light (20g) and have a battery life of +/-5 years, recorded both the normal abdominal temperature of guinea pigs, and their febrile response to an intramuscular injection of 50microg/kg lipopolysaccharide (E . coli) every 5min for the duration of the experiment (21 days), to a resolution of at least 0.05 degrees C . No calibration shifts or loss of data occurred during the study period . However, despite their small size and versatility, we found that the loggers were suited for use only in guinea pigs with a body mass of approximately 600g or greater . In smaller animals, the loggers caused peritonitis. FEBS Lett, 2001 Mar 2, 491(3), 289 - 98 Real time fluorescence analysis of the RecA filament: implications of base pair fluidity in repeat realignment; Sen S et al.; During recombination, when Escherichia coli RecA mediates annealing across DNA repeats, Watson-Crick chemistry can only specify the complementarity of pairing, but not the most optimal frame of alignment . We describe that although stochastic alignments across poly(dA) and poly(dT) can lead to sub-optimally annealed duplexes containing ssDNA gaps/overhangs, the same are realigned into an optimal frame by a putative motor activity of RecA {Sen et al . (2000) Biochemistry 39, 10196-10206} . In the present study, we analyze the nature of realignment intermediates in real time, by employing a fluorescent probe, 2-aminopurine (2AP), which can not only report the status of RecA on the unstacked duplex, but also the fluidity of bases in such a filament . Although known to display a lower affinity for duplex DNA, RecA seems to remain functionally associated with these sub-optimally aligned repeat duplexes, until the realignment approaches completion . Moreover, a comparison of 2AP fluorescence in repeat versus mixed sequences indicates that bases in a RecA repetitive DNA filament exhibit higher degrees of freedom that might mediate a 'non-planar hydrogen bonding cross talk' across the bases on either strand . We discuss a model to explain the mechanistic basis of realignment and its implications in signaling the end of homology maximization, which triggers RecA fall off. FEBS Lett, 2001 Mar 2, 491(3), 169 - 73 Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli; Okuda Y et al.; A magnetosome-associated protein, MAM22, contains a TPR domain (five TPR motifs and one putative TPR motif) that has been known to mediate protein-protein interactions . We expressed the mam22 gene in Escherichia coli and found that the purified MAM22 was reversibly self-aggregated by NaCl . The structural model of MAM22 which has been proposed on the basis of the crystal structure of the N-terminal TPR domain of a human Ser/Thr protein phosphatase suggests the novel hydrophobic colloidal features of MAM22 with TPR motifs. Mutat Res, 2001 Mar 1, 474(1-2), 1 - 14 Fidelity of replication of repetitive DNA in mutS and repair proficient Escherichia coli; Levy DD et al.; Replication fidelity is not constant among strains within a species or at all genetic loci within a genome . Altered fidelity of replication may affect patterns of pathogenesis and the evolution of these strains . We have been studying replication fidelity in Escherichia coli, both in laboratory attenuated strains and in food-borne pathogens . To understand the altered patterns of mutagenesis at the molecular level, we used a shuttle vector plasmid with a tRNA mutational marker gene which had been altered to include homopolymeric runs of five, seven and nine {G:C} pairs, as well as non-repetitive DNA . Replication of the plasmid in mutS strains resulted in a 20-fold increase in mutant progeny plasmids . The mutations were almost all (>90%) frameshift mutations, while base substitution mutations were rare . Most mutations were insertions or deletions of one or two {G:C} pairs in the longest homopolymeric runs . Larger deletions (5 to >70bp), also targeted to the repetitive sequence, were likewise common . Mutations increased exponentially with the length of the homopolymeric run . These patterns of mutation, including unexpectedly high levels in repair proficient strains, led to an examination of the E . coli K-12 genome for homopolymeric DNA . This sequence motif was found to be rare, particularly in genes and open reading frames . Amino acid homotrimers were found to avoid usage of homopolymeric codons, even when they are preferred among synonymous codons in E . coli . There appears to be active selection against tandem direct nucleotide repeats in the E . coli genome, correlated with the inability of the organism to accurately replicate such sequence. Int J Parasitol, 2001 Feb, 31(2), 145 - 53 Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells; de la Fuente J et al.; Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described . The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes . In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined . msp1alpha and msp1beta1 genes from the Oklahoma isolate of A . marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression . Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E . coli . The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E . coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis . Adhesion of the recombinant E . coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy . MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells . In contrast, recombinant E . coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells . When low expression vectors were used, single E . coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E . coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell . With electron microscopy, fusion of E . coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E . coli membranes expressing MSP1a . These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A . marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes . The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts. Int J Parasitol, 2001 Feb, 31(2), 109 - 13 Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I; McLeod R et al.; Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis . It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi . Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain . We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-{2,4 dichlorophenoxy} phenol}), which confirmed all the requisite features of an ENR and its interactions with triclosan . Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P . falciparum and Toxoplasma gondii at low (i.e . IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations . Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds. Eur J Pharmacol, 2001 Mar 2, 414(2-3), 281 - 7 The timing of endothelin and nitric oxide inhibition affects survival in a mice model of septic shock; Iskit AB et al.; The effect of endothelin and nitric oxide (NO) inhibition on survival from septic shock was investigated in male Swiss albino mice (20-40 g), with particular emphasis on the timing of the administration of their blockers after Escherichia coli endotoxin (lipopolysaccharide, O55:B5, 60 mg kg(-1), i.p.) challenge . Mice were injected with the endothelin receptor antagonist bosentan (30 mg kg(-1), i.p., either 2 or 12 h after endotoxin) alone or in addition to the NO synthase blockers L-canavanine (100 mg kg(-1), i.p.), N(G)-nitro-L-arginine methyl ester (L-NAME, 3 mg kg(-1), i.p.) or aminoguanidine (15 mg kg(-1), i.p.), which were also given twice at 2 and 6 h after endotoxin . Control animals received saline, and survival rates in each group (n=10) were recorded over 24 h at 6-h intervals . At 24 h, the survival rate was 10% in controls, but 30% (n.s.) and 70% (P<0.05) in animals that received only bosentan at 2 and 12 h, respectively, indicating a relatively late involvement of endothelin in comparison to NO . In contrast, these figures were 70% (P<0.05) and 80% (P<0.05) at 12 h for L-NAME and L-canavanine, respectively, and 10% (n.s.) at 24 h, implying a relatively early involvement of NO compared to endothelin . Interestingly, survival in the aminoguanidine group (75% at 24 h, P<0.05 vs . controls) was markedly higher than that in the L-NAME and L-canavanine groups, an effect that was attributed to mechanisms other than NO inhibition . Survival was better (60%, P<0.05 vs . endotoxin alone) when bosentan was given at 2 h in combination with L-NAME, but the best outcome (90% survival, P<0.05) was observed in animals when bosentan was given at 12 h and L-NAME was injected twice at 2 and 6 h . However, the statistical analysis revealed no significant additional beneficial effect of L-NAME coadministered with bosentan . Therefore, we conclude that NO is involved during the earlier phases of septic shock in comparison to a relatively late involvement of endothelin peptides, and that bosentan alone appears to be beneficial when administered at least 12 h after the endotoxin challenge in our mice model of septic shock. Biochim Biophys Acta, 2001 Mar 1, 1504(1), 128 - 43 Uncoupling proteins: the issues from a biochemist point of view; Klingenberg M et al.; The functional characteristics of uncoupling proteins (UCP) are reviewed, with the main focus on the results with isolated and reconstituted proteins . UCP1 from brown adipose tissue, the paradigm of the UCP subfamily, is treated in more detail . The issues addressed are the role and mechanism of fatty acids, the nucleotide binding, the regulation by pH and the identification by mutagenesis of residues involved in these functions . The transport and regulatory functions of UCP2 and 3 are reviewed in comparison to UCP1 . The inconsistencies of a proposed nucleotide insensitive H(+) transport by these UCPs as concluded from the expression in yeast and Escherichia coli are elucidated . In both expression system UCP 2 and 3 are not in or cannot be converted to a functionally native state and thus also for these UCPs a nucleotide regulated H (+) transport is postulated. Mol Cell, 2001 Feb, 7(2), 301 - 7 The mechanism of type IA topoisomerase-mediated DNA topological transformations; Li Z et al.; Type IA DNA topoisomerases possess several domains forming a toroidal molecule with a central hole large enough to accommodate single- or double-stranded DNA . The sign inversion model predicts several protein-DNA intermediates, including those in which DNA is trapped within the hole . Opposing cysteine residues were incorporated into two independent domains surrounding the putative DNA binding cavity of E . coli topoisomerase III, creating a molecule that can be covalently closed or opened by oxidizing or reducing the disulfide bond . The formation of the disulfide bond allowed the trapping of single- and double-stranded DNA within the cavity of the enzyme and the identification of other intermediates proposed by the sign inversion model. Cell, 2001 Feb 9, 104(3), 433 - 40 A novel all helix fold of the AP180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis; Mao Y et al.; Clathrin-mediated endocytosis plays a major role in retrieving synaptic vesicles from the plasma membrane following exocytosis . This endocytic process requires AP180 (or a homolog), which promotes the assembly and restricts the size of clathrin-coated vesicles . The highly conserved 33 kDa amino-terminal domain of AP180 plays a critical role in binding to phosphoinositides and in regulating the clathrin assembly activity of AP180 . The crystal structure of the amino-terminal domain reported herein reveals a novel fold consisting of a large double layer of sheets of ten alpha helices and a unique site for binding phosphoinositides . The finding that the clathrin-box motif is most