Escherichia coli

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 mostly buried and lies in a helix indicates a different site and mechanism for binding of the domain to clathrins than previously assumed.

Microbiology, 2001 Mar, 147(Pt 3), 709 - 15
Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes; Castanie-Cornet MP et al.; Acid resistance is an important feature of both pathogenic and non-pathogenic Escherichia coli . It enables survival in the acidic regions of mammalian gastrointestinal tracts and is largely responsible for the small number of bacteria required for infection/colonization . Three systems of acid resistance have been identified, the most efficient of which requires glutamic acid during pH 2 acid challenge . Three proteins associated with glutamate-dependent acid resistance have been identified . They are glutamate decarboxylase (encompassing two isozymes encoded by gadA and gadB) and a putative glutamate:gamma-amino butyric acid antiporter (encoded by gadC) . The results confirm that the GadA and GadB proteins increase in response to stationary phase and low environmental pH . The levels of these proteins correspond to concomitant changes in gadA and gadBC mRNA levels . Fusions between lacZ and the gadA and gadBC operons indicate that this control occurs at the transcriptional level . Western blot, Northern blot and fusion analyses reveal that regulation of these genes is complex . Expression in rich media is restricted to stationary phase . However, in minimal media, acid pH alone can trigger induction in exponential or stationary phase cells . Despite this differential control, there is only one transcriptional start site for each gene . Expression in rich media is largely dependent on the alternate sigma factor sigma(S) and is repressed by the cAMP receptor protein (CRP) . In contrast, sigma(S) has only a minor role in gad transcription in cells grown in minimal media . Deletions of the regulatory region upstream of gadA provided evidence that a 20 bp conserved region located 50 bp from the transcriptional start of both operons is required for expression.

Microbiology, 2001 Mar, 147(Pt 3), 571 - 80
An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas; Blomquist CL et al.; Membrane proteins mediate several important processes, including attachment, in several Mollicute species . Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors . Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission . A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced . PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots . This PCR product was used to identify a 2.5 kbp ECO:RI-HIN:dIII fragment that was cloned and sequenced . A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers . Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point . The amino acid sequence had no significant homology with other known phytoplasma IDP genes . The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum . A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E . coli-expressed and native WX phytoplasma proteins . This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.

Microbiology, 2001 Mar, 147(Pt 3), 561 - 70
NAD(P)H:menadione oxidoreductase of the amitochondriate eukaryote Giardia lamblia: a simpler homologue of the vertebrate enzyme; Sanchez LB et al.; The amitochondriate eukaryote Giardia lamblia contains an NAD(P)H:menadione oxidoreductase (EC 1.6.99.2) (glQR) that catalyses the two-electron transfer oxidation of NAD(P)H with a quinone as acceptor . The gene encoding this protein in G . lamblia was expressed in Escherichia coli . The purified recombinant protein had an NAD(P)H oxidoreductase activity, with NADPH being a more efficient electron donor than NADH . Menadione, naphthoquinone and several artificial electron acceptors served as substrate for the enzyme . glQR shows high amino acid similarity to its homologues in vertebrates and also to a series of hypothetical proteins from bacteria . Although glQR is considerably smaller than the mammalian enzymes, three-dimensional modelling shows similar arrangement of the secondary structural elements . Most amino acid residues of the mammalian enzymes that participate in substrate binding or catalysis are conserved . Conservation of these features and the similarity in substrate specificity and in susceptibility to inhibitors establish glQR as an authentic member of this protein family.

Mol Cell Biol, 2001 Mar, 21(5), 1737 - 46
TFIIA interacts with TFIID via association with TATA-binding protein and TAF40; Kraemer SM et al.; TFIIA and TATA-binding protein (TBP) associate directly at the TATA element of genes transcribed by RNA polymerase II . In vivo, TBP is complexed with approximately 14 TBP-associated factors (TAFs) to form the general transcription factor TFIID . How TFIIA and TFIID communicate is not well understood . We show that in addition to making direct contacts with TBP, yeast TAF40 interacts directly and specifically with TFIIA . Mutational analyses of the Toa2 subunit of TFIIA indicate that loss of functional interaction between TFIIA and TAF40 results in conditional growth phenotypes and defects in transcription . These results demonstrate that the TFIIA-TAF40 interaction is important in vivo and indicate a functional role for TAF40 as a bridging factor between TFIIA and TFIID.

Mol Cell Biol, 2001 Mar, 21(5), 1688 - 99
Ambient pH signaling regulates nuclear localization of the Aspergillus nidulans PacC transcription factor; Mingot JM et al.; The Aspergillus nidulans zinc finger transcription factor PacC is activated by proteolytic processing in response to ambient alkaline pH . The pH-regulated step is the transition of full-length PacC from a closed to an open, protease-accessible conformation . Here we show that in the absence of ambient pH signaling, the C-terminal negative-acting domain prevents the nuclear localization of full-length closed PacC . In contrast, the processed PacC form is almost exclusively nuclear at any ambient pH . In the presence of ambient pH signaling, the fraction of PacC that is in the open conformation but has not yet been processed localizes to the nucleus . Therefore, ambient alkaline pH leads to an increase in nuclear PacC by promoting the proteolytic elimination of the negative-acting domain to yield the processed form and by increasing the proportion of full-length protein that is in the open conformation . These findings explain why mutations resulting in commitment of PacC to processing irrespective of ambient pH lead to permanent PacC activation and alkalinity mimicry . A nuclear import signal that targets Escherichia coli beta-galactosidase to the nucleus has been located to the PacC zinc finger region . A mutation abolishing DNA binding does not prevent nuclear localization of the processed form, showing that PacC processing does not lead to nuclear localization by passive diffusion of the protein made possible by the reduction in size, followed by retention in the nucleus after DNA binding.

Mol Cell Biol, 2001 Mar, 21(5), 1463 - 74
Autoinhibition mechanism of proto-Dbl; Bi F et al.; The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family . Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues . The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function . Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences . Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential . Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain . Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity . The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain . Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.

J Neurochem, 2001 Mar, 76(5), 1315 - 25
Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis; Strong MJ et al.; The intraneuronal aggregation of phosphorylated high-molecular-weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS) . In order to determine whether this observation is due to the aberrant or hyper-phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls . We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X-100 insoluble protein fractions, with respect to migration patterns on 2D-iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain-mediated proteolysis . The rate of calpain-mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction . Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples . Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.

J Immunol, 2001 Mar 15, 166(6), 3655 - 8
Cutting edge: human gamma delta T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis; Altincicek B et al.; Activation of V gamma 9/V delta 2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites . We suggested earlier that compounds synthesized by the 2-C:-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the V gamma 9/V delta 2 T cell reactivity of many pathogens . Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E . coli extracts to stimulate gamma delta T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.

J Clin Invest, 2001 Mar, 107(5), 621 - 9
Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function; McNamara BP et al.; The mechanisms by which enteropathogenic Escherichia coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined . EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells . Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system . We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells . However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin . Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy . These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

Genes Dev, 2001 Mar 1, 15(5), 627 - 37
The RssB response regulator directly targets sigma(S) for degradation by ClpXP; Zhou Y et al.; The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes . Control over sigma(S) activity is exercised in part by regulated degradation of sigma(S) . In vivo, degradation requires the ClpXP protease together with RssB, a protein homologous to response regulator proteins . Using purified components, we reconstructed the degradation of sigma(S) in vitro and demonstrate a direct role for RssB in delivering sigma(S) to ClpXP . RssB greatly stimulates sigma(S) degradation by ClpXP . Acetyl phosphate, which phosphorylates RssB, is required . RssB participates in multiple rounds of sigma(S) degradation, demonstrating its catalytic role . RssB promotes sigma(S) degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP . sigma(S) and RssB form a stable complex in the presence of acetyl phosphate, and together they form a ternary complex with ClpX that is stabilized by ATP{gamma-S} . Alone, neither sigma(S) nor RssB binds ClpX with high affinity . When ClpP is present, a larger sigma(S)--RssB--ClpXP complex forms . The complex degrades sigma(S) and releases RssB from ClpXP in an ATP-dependent reaction . Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.

Clin Chem, 2001 Mar, 47(3), 471 - 6
Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB; Sunahara Y et al.; BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury . We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum . METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli . The resulting three types of CK isoenzymes were purified by conventional chromatography . RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity . Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells . Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB . Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB . The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L . CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

Clin Diagn Lab Immunol, 2001 Mar, 8(2), 424 - 8
Dose-dependent circulating immunoglobulin A antibody-secreting cell and serum antibody responses in Swedish volunteers to an oral inactivated enterotoxigenic Escherichia coli vaccine; Jertborn M et al.; The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection . The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs) . Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given . The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine . Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4 . The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria . These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.

Clin Diagn Lab Immunol, 2001 Mar, 8(2), 283 - 7
Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity; Zheng L et al.; Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically . A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies . Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further . Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein . The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses . However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV . Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein . This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.

Bioinformatics, 2001 Feb, 17(2), 155 - 61
Automated extraction of information on protein-protein interactions from the biological literature; Ono T et al.; MOTIVATION: To understand biological process, we must clarify how proteins interact with each other . However, since information about protein-protein interactions still exists primarily in the scientific literature, it is not accessible in a computer-readable format . Efficient processing of large amounts of interactions therefore needs an intelligent information extraction method . Our aim is to develop an efficient method for extracting information on protein-protein interaction from scientific literature . RESULTS: We present a method for extracting information on protein-protein interactions from the scientific literature . This method, which employs only a protein name dictionary, surface clues on word patterns and simple part-of-speech rules, achieved high recall and precision rates for yeast (recall = 86.8% and precision = 94.3%) and Escherichia coli (recall = 82.5% and precision = 93.5%) . The result of extraction suggests that our method should be applicable to any species for which a protein name dictionary is constructed . AVAILABILITY: The program is available on request from the authors.

Biochem J, 2001 Mar 15, 354(Pt 3), 663 - 70
Substrate-binding characteristics of proteins in the 90 kDa heat shock protein family; Nemoto TK et al.; In the present study we investigated the substrate-binding characteristics of three members of the 90 kDa heat shock protein (HSP90) family, namely the alpha isoform of human HSP90 (HSP90alpha), human GRP94 (94 kDa glucose-regulated protein, a form of HSP90 from endoplasmic reticulum), and HtpG (the Escherichia coli homologue of HSP90) and the domain responsible for these characteristics . The recombinant forms of HSP90alpha, GRP94 and HtpG existed as dimers and became oligomerized at higher temperatures . Among the three family members, HtpG required the highest temperature (65 degrees C) for its transition to oligomeric forms . The precipitation of the substrate protein, glutathione S-transferase, which occurred at 55 degrees C, was efficiently prevented by the simultaneous presence of a sufficient amount of HSP90alpha or GRP94, but not by HtpG, which was still present as a dimer at that temperature . However, precipitation was stopped completely at 65-70 degrees C, at which temperature HtpG was oligomerized . Thus the transition of HSP90-family proteins to a state with self-oligomerization ability is essential for preventing the precipitation of substrate proteins . We then investigated the domain responsible for the substrate binding of HtpG on the basis of the three domain structures . The self-oligomerizing and substrate-binding activities towards glutathione S-transferase and citrate synthase were both located in a single domain, the N-terminal domain (residues 1-336) of HtpG . We therefore propose that the primary peptide-binding site is located in the N-terminal domain of HSP90-family proteins.

Biochem J, 2001 Mar 15, 354(Pt 3), 627 - 33
Affinity of mismatch-binding protein MutS for heteroduplexes containing different mismatches; Brown J et al.; We have used bandshift analysis to measure the interaction between the Escherichia coli mismatch-binding protein MutS and synthetic DNA fragments containing all possible DNA mismatches as well as an unpaired T (DeltaT) . The order of affinity is found to be DeltaT>GT>GG>AA approximately TT approximately TC>CA>GA>CC>GC . We find that the affinity for GT mismatches is affected by the flanking sequence and decreases in the order G(n)C(n)>(GC)(n)>A(n)T(n)>(AT)(n) . Studies with base analogues show good binding to varphiT (where varphi represents 1',2'-dideoxyribose), but much weaker binding to Gvarphi.

Biochem J, 2001 Mar 15, 354(Pt 3), 553 - 9
The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands; Klappa P et al.; Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes . In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp . Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp . To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.

Biochem J, 2001 Mar 15, 354(Pt 3), 521 - 9
Expression pattern and localization of beta,beta-carotene 15,15'-dioxygenase in different tissues; Wyss A et al.; Beta,beta-carotene 15,15'-dioxygenase cleaves beta,beta-carotene into two molecules of retinal, and is the key enzyme in the metabolism of beta,beta-carotene to vitamin A . The enzyme has been known for more than 40 years, yet all attempts to purify the protein to homogeneity have failed . Recently, the successful cloning and sequencing of an enzyme with beta,beta-carotene 15,15'-dioxygenase activity from chicken, as well as from Drosophila, has been reported . Here, we describe in detail our attempt to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent as to allow determination of partial amino acid sequences, which were then used to design degenerate oligonucleotides . Screening of a chicken duodenal expression library yielded a full-length clone containing a coding sequence of 1578 bp . Functional expression in Escherichia coli and in eukaryotic cell lines confirmed that we had cloned the first vertebrate dioxygenase that cleaves beta,beta-carotene at the central 15,15'-double bond . By performing a sequence homology search, the cDNA sequence of the mouse homologue was found as an expressed sequence tag (EST) in the gene bank . At the amino-acid level, the degree of homology between the chicken and mouse sequences is 81% . Thus beta,beta-carotene 15,15'-dioxygenase can be considered as being an enzyme that is evolutionarily rather well conserved . We established the expression pattern of beta,beta-carotene 15,15'-dioxygenase in chicken and mouse tissues with a combination of Northern blots and in situ hybridization . The mRNA for beta,beta-carotene 15,15'-dioxygenase was localized primarily in duodenal villi, as well as in liver and in tubular structures of lung and kidney . These new findings demonstrate that beta,beta-carotene 15,15'-dioxygenase is also expressed in epithelial structures, where it serves to provide the tissue-specific vitamin A supply.

J Infect Dis, 2001 Apr 1, 183(7), 1071 - 8 Epub 2001 Mar 08.
Further characterization of Escherichia coli brain microvascular endothelial cell invasion gene ibeA by deletion, complementation, and protein expression; Huang SH et al.; The ibeA gene (ibe10) previously identified by TnphoA mutagenesis is part of a 50-kDa full-length open-reading frame (ORF) encoded by a 1.37-kb DNA fragment . An isogenic in-frame deletion mutant of ibeA (ZD1) was constructed by chromosomal gene replacement with a suicide plasmid pCVD442 carrying a 2.1-kb DNA fragment with an ibeA deletion . Similar to the previously described TnphoA insertion mutant of ibeA, the isogenic ibeA deletion mutant ZD1 was significantly less invasive in human brain microvascular endothelial cells (BMECs) than the parent strain . The mutant ZD1 was fully complemented by the ibeA ORF . The ibeA gene was subcloned into pET28a(+) and was expressed as a recombinant protein with an N-terminal histidine tag . The recombinant IbeA protein had much greater activity (50 times) in blocking the invasion of BMECs by Escherichia coli K1 than did the partial protein fragment, which provides further evidence that ibeA is an important determinant for E . coli K1 invasion of BMECs.

Biochem Biophys Res Commun, 2001 Mar 9, 281(4), 993 - 1000
Metabolic biotinylation of secreted and cell surface proteins from mammalian cells; Parrott MB et al.; Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of medical and scientific applications ranging from drug targeting to immunohistochemistry . To maximize the application of this technology in mammalian systems, we recently demonstrated the ability to metabolically biotinylate tagged proteins in mammalian cells using the endogenous biotin ligase enzymes of the mammalian cell . This technology allows site-specific biotinylation without any exogenous reagents and eliminates possible inactivation of the protein of interest by nonspecific biotinylation . Here, we report further expansion of the mammalian metabolic biotinylation technology to enable biotinylation of proteins secreted from mammalian cells and expressed on their cell surface by cosecretion with BirA, the biotin ligase of E . coli . This technique can be used to biotinylate secreted proteins for purification or targeting and also for biotinylating the surfaces of mammalian cells to facilitate their labeling and purification from other nontagged cells .

Biochem Biophys Res Commun, 2001 Mar 9, 281(4), 962 - 5
Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli; Lorenzo LJ et al.; Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins . Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract . In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate . SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120 . The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation . Spherical structures with an average diameter of 30 nm were observed using electron microscopy . We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E . coli .

Biochem Biophys Res Commun, 2001 Mar 2, 281(3), 810 - 4
A novel single nucleotide polymorphism altering stability and activity of CYP2a6; Ariyoshi N et al.; CYP2A6 is known as a major cytochrome P450 (CYP) responsible for the oxidation of nicotine and coumarin in humans . In this study, we explored genetic polymorphisms, which reduce CYP2A6 activity in Japanese . Two novel mutations in exon 9 of the CYP2A6 gene were found . A single nucleotide polymorphism of T1412C and G1454T resulted in Ile471Thr and Arg485Leu substitution, respectively . The frequency of the former variant allele was considerably high (15.7%), while the latter variant appeared to be a rare polymorphism . Heterologous expression of CYP2A6 using a cDNA possessing C instead of T-base at codon 471 in Escherichia coli caused remarkable reduction of the stability of holoenzyme at 37 degrees C . Furthermore, this variant enzyme almost lacked nicotine C-oxidase activity, although coumarin 7-hydroxylase activity was still observed . These data suggest that individuals homozygous for the T1412C variant allele or heterozygous for this and a defect allele such as the CYP2A6*4 may be poor metabolizer of nicotine, but not coumarin .

Biochem Biophys Res Commun, 2001 Mar 2, 281(3), 783 - 7
Investigation of subunit function in ADP-glucose pyrophosphorylase; Kavakli IH et al.; ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (alpha(2)beta(2)) . Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles . To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain . An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions . Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus . This single mutation had no effect on the Km for ATP and Mg(2+), which were similar to the WT enzyme . The K(m) for glucose 1-P, however, was sixfold higher than the WT enzyme . These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS .

Protein Expr Purif, 2001 Mar, 21(2), 361 - 5
Purification of a fragment of MDM2 for production of polyclonal antisera; Carstens BP et al.; A histidine-tagged, carboxy-terminal fragment of the murine double minute 2 gene product, p90(MDM2), was purified by Ni--NTA chromatography and preparative gel electrophoresis . The purified MDM2 fragment was used to generate polyclonal antisera that recognize multiple species of MDM2 proteins, including the inhibitor of p53, p90(MDM2), as well as the activator of p53, p76(MDM2) . The antibodies are useful for Western analysis, immunoprecipitation, and immunofluorescence .

Protein Expr Purif, 2001 Mar, 21(2), 352 - 60
Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography; Owens RM et al.; One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B . Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene . Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II) . In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins .

Protein Expr Purif, 2001 Mar, 21(2), 317 - 22
Expression, purification, and characterization of the active immunoglobulin-like domain of human granulocyte-colony-stimulating factor receptor in Escherichia coli; Ishibashi M et al.; We succeeded in the expression, purification, and refolding of the immunoglobulin-like (Ig) domain of human granulocyte-colony-stimulating factor (G-CSF) receptor with amino-terminal His-tag in Escherichia coli . The refolded Ig domain bound to a G-CSF affinity column and could be eluted with free G-CSF as a receptor-ligand complex, demonstrating that the Ig domain has the information necessary for binding its ligand, G-CSF . The eluted His-Ig/G-CSF complex could be separated from excess G-CSF by Ni-NTA column chromatography . The yield of this active recombinant His-Ig protein is about 0.72 mg per liter of culture . Its small size and the ease of production make this receptor fragment a useful reagent for the structural analysis of its complex with G-CSF .

Protein Expr Purif, 2001 Mar, 21(2), 303 - 9
Overexpression and purification of the membrane-bound cytochrome P450 2B4; Saribas AS et al.; Expression of the membrane-bound cytochrome P450 2B4 by the pLW01-P450 expression vector, which utilizes a T7 promoter, is markedly improved by employing Escherichia coli strain C41(DE3) {Miroux, B., and Walker, J . (1996) J . Mol . Biol 260, 289--298; Bridges, A., Gruenke, L., Chang, Y.-T., Vasker, I., Loew, G., and Waskell, L . (1998) J . Biol . Chem . 273, 17036--17049} . Using this expression system, it was possible to routinely obtain an average of 50--60 mg and as high as 100 mg of cyt P450 2B4 per liter of cell culture in volumes of 500 ml . An improved purification procedure for cyt P450 2B4 is also described which allows recovery of 30% of the expressed protein . It was possible in one step using B-PER reagent and polyoxyethylene-9-lauryl ether to both lyse the E . coli and solubilize the expressed cyt P450 . Cyt P450 2B4 with a specific content of 17 nmol/mg protein and a single band on polyacrylamide gel electrophoresis was routinely isolated . The yield of cyt P450 from the improved purification procedure is twice that from the original procedure and the purity of the recovered protein typically has a specific content of 17 nmol cyt P450/mg of protein .

Protein Expr Purif, 2001 Mar, 21(2), 275 - 85
Expression and glycosylation studies of human FGF receptor 4; Tuominen H et al.; Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs . FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design . Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies . We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated . The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous . Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E . coli-produced FGFR4(ed) still aggregates .

Protein Expr Purif, 2001 Mar, 21(2), 268 - 74
Cloning, expression, purification, and characterization of rat MMP-12; Fu JY et al.; Macrophage metalloelastase (MMP-12) is implicated in the pathology of many diseases such as emphysema, aortic lesions and cancer . Recently, MMP-12 was cloned and purified from mouse and human macrophages . We report here the expression of the full-length and catalytic domain of rat MMP-12 in Escherichia coli and characterization of the purified enzyme . Inclusion bodies of expressed rat MMP-12 catalytic domain were denatured and refolded using a new method, and then affinity purified to near homogeneity with zinc-chelating Sepharose . The purified rat MMP-12 catalytic domain was highly active in digesting substrates, having a K(m) of 12 microM and optimal pH of 7.5--8.5 . During investigation of natural substrate specificity, we found that rat MMP-12 catalytic domain was able to completely degrade collagen-V, partially degrade collagen-I, but it was unable to digest collagen-IV . The enzyme could also degrade osteonectin, vitronectin, and fibronectin, but not laminin and albumin . The catalytic properties and natural substrate specificity of rat MMP-12 catalytic domain differed from those of human MMP-12 catalytic domain .

Protein Expr Purif, 2001 Mar, 21(2), 260 - 7
Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification; Goenka S et al.; zeta-Crystallin is a taxon-specific crystallin found in the eye lens of guinea pig and other hystricomorph rodents and camelids . It is an NADPH:quinone oxidoreductase and is also present in low amounts in other tissues where it might act as a detoxifying enzyme . A lens-specific promoter confers lens-specific expression of the gene in high amounts where it is speculated to play a structural role in maintaining the transparency of the lens ensemble . A deletion mutation leads to autosomal dominant congenital cataract and also results in the loss of NADPH binding . In order to perform structural studies with the protein with an aim to delineate the cause of cataract in these mutant guinea pigs, recombinant zeta-crystallin was cloned and expressed in Escherichia coli . The overexpression of the protein in E . coli resulted in a major fraction of it partitioning into inclusion bodies . The co-overexpression of the bacterial chaperone system GroEL/ES along with zeta-crystallin could significantly enhance the yield of soluble protein . Active zeta-crystallin could then be purified from the E . coli using Mono Q anion exchange FPLC and was found to be identical to the native zeta-crystallin isolated from the guinea pig lens with respect to size, spectral properties, and activity .

Mol Ther, 2001 Feb, 3(2), 233 - 40
Combined adenovirus-mediated nitroreductase gene delivery and CB1954 treatment: a well-tolerated therapy for established solid tumors; Djeha AH et al.; Gene-directed enzyme prodrug therapy (GDEPT) is a refinement of cancer chemotherapy that generates a potent cell-killing drug specifically in tumor cells by enzymatic activation of an inert prodrug . We describe in vivo studies that evaluate the efficacy and safety of intratumoral (i.t.) injection of an adenovirus vector (CTL102) expressing Escherichia coli nitroreductase (NTR) combined with systemic prodrug (CB1954) treatment . A single i.t . injection of CTL102 (7.5 x 10(9) to -2 x 10(10) particles) followed by CB1954 treatment produced clear anti-tumor effects in subcutaneous (s.c.) xenograft models of four cancers that are likely candidates for GDEPT (i.e., primary liver, head and neck, colorectal and prostate) . Virus dose-response studies (s.c . liver model) revealed a steep increase and subsequent rapid plateauing of both NTR gene delivery and anti-tumor efficacy . Evidence of minor virus spread (toxicity) was observed in a s.c . head and neck xenograft model . This was eliminated by passive immunization with neutralizing anti-Ad5 antibodies prior to virus injection without reducing the magnitude of the anti-tumor effect . Preexisting anti-Ad5 neutralizing antibodies may therefore be an advantage rather than an issue in the clinical use of this new therapy.

Mol Ther, 2001 Feb, 3(2), 206 - 15
Host responses and persistence of vector genome following intrabronchial administration of an E1(-)E3(-) adenovirus gene transfer vector to normal individuals; Harvey BG et al.; Adenovirus (Ad)-mediated gene transfer to the respiratory epithelium of experimental animals and to nasal and airway epithelium of individuals with cystic fibrosis is followed by transient gene expression . Extensive studies in experimental animals are consistent with the concept that local cellular host anti-vector immune responses account for this short-term expression, and systemic and local {lung epithelial lining fluid (ELF)} anti-Ad neutralizing antibodies are generated following Ad vector administration to the respiratory epithelial surface . To determine if this paradigm holds in normal humans, a first-generation Ad vector (Ad(GV)CD.10, an E1(-)E3(-) Ad serotype 5-based vector coding for the Escherichia coli cytosine deaminase gene) was sprayed locally in escalating doses (8 x 10(8)-8 x 10(10) particle units (pu), n = 2/group) into the lung airway epithelium of six normal individuals . Serum, ELF, and endobronchial biopsies were obtained at baseline and at various time points following vector administration . In contrast to the observations in experimental animals in which lung administration of first-generation Ad vectors is followed by strong systemic and local host response, bronchial spray administration of the Ad vector to normal humans showed: (1) minimal inflammation in bronchial biopsies, bronchial brushing, and bronchoalveolar lavage fluid; (2) no blood lymphocyte proliferation in five of six individuals in response to in vitro stimulation with Ad antigens; and (3) no significant increase from baseline in blood or lung ELF anti-Ad neutralizing antibodies . Despite this minimal normal human anti-Ad host response, dose-dependent levels of vector DNA in the airway epithelium were transient . Vector DNA in the targeted airway epithelial cells peaked in a dose-dependent fashion at 0.007 to 1.1 copies/cell at day 7 and declined thereafter, reducing to <10% of peak levels by 2 weeks . These observations demonstrate both the strengths and the limits of using experimental animals to predict human responses to gene transfer vectors . While the transient nature of Ad vector persistence in the airway epithelium is predicted by most experimental animal studies, respiratory epithelial administration of first-generation Ad vectors at doses up to 10(10) pu to airway epithelium of healthy individuals elicits minimal to no detectable systemic and mucosal humoral and cellular immune responses, an observation diametrically opposed to the host responses measured in experimental animals . These findings suggest that, while adaptive anti-Ad immune responses likely play some role in the disappearance of the vector DNA following vector administration to the human lung, other mechanisms may also be involved in the response of humans to Ad gene transfer vectors.

J Mol Biol, 2001 Mar 9, 306(5), 1115 - 26
Crystal structures of the maltodextrin/maltose-binding protein complexed with reduced oligosaccharides: flexibility of tertiary structure and ligand binding; Duan X et al.; The structure of the maltodextrin or maltose-binding protein, an initial receptor for bacterial ABC-type active transport and chemotaxis, consists of two globular domains that are separated by a groove wherein the ligand is bound and enclosed by an inter-domain rotation . Here, we report the determination of the crystal structures of the protein complexed with reduced maltooligosaccharides (maltotriitol and maltotetraitol) in both the "closed" and "open" forms . Although these modified sugars bind to the receptor, they are not transported by the wild-type transporter . In the closed structures, the reduced sugars are buried in the groove and bound by both domains, one domain mainly by hydrogen-bonding interactions and the other domain primarily by non-polar interactions with aromatic side-chains . In the open structures, which abrogate both cellular activities of active transport and chemotaxis because of the large separation between the two domains, the sugars are bound almost exclusively to the domain rich in aromatic residues . The binding site for the open chain glucitol residue extends to a subsite that is distinct from those for the glucose residues that were uncovered in prior structural studies of the binding of active linear maltooligosaccharides . Occupation of this subsite may also account for the inability of the reduced oligosaccharides to be transported . The structures reported here, combined with those previously determined for several other complexes with active oligosaccharides in the closed form and with cyclodextrin in the open form, revealed at least four distinct modes of ligand binding but with only one being functionally active . This versatility reflects the flexibility of the protein, from very large motions of interdomain rotation to more localized side-chain conformational changes, and adaptation by the oligosaccharides as well.

J Mol Biol, 2001 Mar 9, 306(5), 997 - 1010
Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference; Jeffery DA et al.; As part of a nutrient-responsive signaling pathway, the budding yeast cyclin-CDK complex Pho80-Pho85 phosphorylates the transcription factor Pho4 on five sites and inactivates it . Here, we describe the kinetic reaction between Pho80-Pho85 and Pho4 . Through experimentation and computer modeling we have determined that Pho80-Pho85 phosphorylates Pho4 in a semi-processive fashion that results from a balance between kcat and k(off) . In addition, we show that Pho80-Pho85 phosphorylates certain sites preferentially . Phosphorylation of the site with the highest preference inhibits the transcriptional activity of Pho4 when it is in the nucleus, while phosphorylation of the lowest-preference sites is required for export of Pho4 from the nucleus . This method of phosphorylation may allow Pho80-Pho85 to quickly inactivate Pho4 in the nucleus and efficiently phosphorylate Pho4 to completion.

J Mol Biol, 2001 Mar 9, 306(5), 969 - 84
Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage; Williams NL et al.; DNA gyrase catalyses DNA supercoiling by passing one segment of DNA (the T segment) through another (the G segment) in a reaction coupled to the binding and hydrolysis of ATP . The N-terminal domains of the gyrase B dimer constitute an ATP-operated clamp that is proposed to capture the T segment during the DNA supercoiling reaction . We have locked this clamp in the closed conformation using the non-hydrolysable ATP analogue ADPNP (5'-adenylyl beta,gamma-imidodiphosphate) . The clamp-locked enzyme is able to bind and cleave DNA, albeit at a reduced level . Although the locked enzyme is not capable of carrying out DNA supercoiling, it can catalyse limited DNA relaxation, consistent with the ability to complete one strand passage event per enzyme molecule via entry of the T segment through the exit gate of the enzyme . The DNA-protein complex of the clamp-locked enzyme has a conformation that differs from the normal positively wrapped conformation of the gyrase-DNA complex . These experiments confirm the role of the ATP-operated clamp in the strand-passage reactions of gyrase and suggest a model for the interaction of DNA with gyrase in which a conformation with the T segment in equilibrium across the DNA gate can be achieved via T-segment entry through the ATP-operated clamp or through the exit gate.

J Mol Biol, 2001 Mar 9, 306(5), 945 - 55
Monomer/dimer ratios of replication protein modulate the DNA strand-opening in a replication origin; Kruger R et al.; DNA opening is an essential step in the initiation of replication via the Cairns mode of replication . The opening reaction was investigated in a gamma ori system by using hyperactive variants of plasmid R6K-encoded initiator protein, pi . Reactivity to KMnO4 (indicative of opening) within gamma ori DNA occurred in both strands of a superhelical template upon the combined addition of wt pi, DnaA and integration host factor (IHF), each protein known to specifically bind gamma ori . IHF, examined singly, enhanced reactivity to KMnO4 . The IHF-dependent reactive residues, however, are distinct from those dependent on pi (wt and hyperactive variants) . Remarkably, the DNA helix opening does not require IHF and/or DnaA when hyperactive variants of pi were used instead of wt protein . We present three lines of evidence consistent with the hypothesis that DNA strand separation is facilitated by pi monomers despite the fact that both monomers and dimers of the protein can bind to iterons (pi binding sites) . Taken together, our data suggest that pi elicits its ability to modulate plasmid copy number at the DNA helix-opening step.

J Mol Biol, 2001 Feb 16, 306(2), 307 - 27
Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations . Application to amino acid recognition by aspartyl-tRNA synthetase; Archontis G et al.; Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code . Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vivo . In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations . Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort . Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase . A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities . The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design.The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the "Asp-like" orientation studied earlier . The new orientation is probably unsuitable for catalysis . A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase . It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding . Insight into the origin of these changes is provided by the component analyses . The double mutation (K198L,D233E) has a similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly . No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here . In molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 A compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket . These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes . Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis.

J Mol Biol, 2001 Feb 16, 306(2), 263 - 74
Identification of the domain in the human interleukin-11 receptor that mediates ligand binding; Schleinkofer K et al.; The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily . The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130 . The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3 . The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3) . We investigated the structural and functional role of the third extracellular receptor domain of IL-11R . A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding . Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11 . To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified . For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used . By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor . These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.

Cytokine, 2001 Feb 21, 13(4), 209 - 19
Analysis of eosinophils and myeloid progenitor responses to modified forms of MPIF-2; Grzegorzewski KJ et al.; Myeloid progenitor inhibitory factor (MPIF)-2 is a beta-chemokine with select and potent activities on eosinophils and myeloid progenitors . In the beta-chemokine family, biological activity is modulated by differential processing of the amino-terminus . Here, for MPIF-2, we describe the biological activities of NH(2)-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities . Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors . For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity . One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays . Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation . In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities .

Anal Biochem, 2001 Mar, 290(2), 359 - 65
Enzymatic determination of homocysteine in cell extracts; Fu TF et al.; Determination of homocysteine levels in cells and serum is important because high homocysteine is a risk factor for cardiovascular disease . The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment . Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer . The reproducibility of the assay is consistent with the other methods currently used . A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter . Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase . The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample . In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP(+) . In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent . L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response .

Anal Biochem, 2001 Mar, 290(2), 205 - 13
Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays; Wendisch VF et al.; Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini . We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E . coli poly(A) polymerase I and purifying it by oligo(dT) chromatography . Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon . More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E . coli genome . Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA . These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E . coli mRNAs .

Anal Biochem, 2001 Mar, 290(2), 186 - 204
Recombinant phycobiliproteins . Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains; Cai YA et al.; A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp . PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag . Such tagged alpha or beta subunits of Anabaena sp . PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin . All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome . The C-terminal 114-residue portion of the Anabaena sp . PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp . His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp . were >30% biotinylated . In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads . Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation .

J Inorg Biochem, 2001 Jan 15, 83(2-3), 115 - 9
The EPR spectrum for CuB in cytochrome c oxidase; Pezeshk A et al.; Incubation of cytochrome c oxidase (CcO) in its resting state in saturated ammonium sulfate, at room temperature overnight, gave EPR signals characteristic of a single Cu(II) center . From the g// and A// values it is concluded that this is a square-planar type 2 copper center, and superhyperfine splitting shows the presence of three nearly equivalent 14N nuclei in the plane . It is suggested that this center, also formed by incubating the enzyme in 10% methanol followed by direct irradiation, must be the CuB center . This type 2 copper EPR spectrum is identical to the EPR spectrum of CuB reported for the isolated cytochrome bo3 complex from Escherichia coli; and to the EPR spectrum reported for the sulfobetaine 12 heat-treated cytochrome c oxidase complex . It is argued that a small perturbation in the system causes decoupling of the magnetic coupling of the heme a3-CuB binuclear center and the appearance of the type 2 EPR signal.

Indian J Pediatr, 2001 Jan, 68(1), 91 - 3
Asymptomatic neonatal cholelithiasis; Citak EC et al.; Cholelithiasis in neonates and infants has been rarely reported . With the current widespread use of diagnostic ultrasonography, more neonates may be found with gallstones and common bile duct stones . We describe a case of asymptomatic gallstones detected incidentally at the age of four days who presented with early onset of neonatal sepsis and dehydration.

Nature, 2001 Feb 15, 409(6822), 947 - 8
A physical map of human chromosome 14; Bruls T et al.; We report the construction of a tiling path of around 650 clones covering more than 99% of human chromosome 14 . Clone overlap information to assemble the map was derived by comparing fully sequenced clones with a database of clone end sequences (sequence tag connector strategy) . We selected homogeneously distributed seed points using an auxiliary high-resolution radiation hybrid map comprising 1,895 distinct positions . The high long-range continuity and low redundancy of the tiling path indicates that the sequence tag connector approach compares favourably with alternative mapping strategies.

Int J Radiat Biol, 2001 Feb, 77(2), 155 - 64
DNA strand break yields after post-high LET irradiation incubation with endonuclease-III and evidence for hydroxyl radical clustering; Milligan JR et al.; PURPOSE: To determine the increase in single- (SSB) and double-strand break (DSB) yields after post-high LET irradiation incubation of plasmid DNA with the endonuclease-III (endo-III) of Escherichia coli . MATERIALS AND METHODS: Plasmid DNA in aerobic aqueous solution was irradiated with one of five radiation types: 137Cs gamma-rays (LET approximately 0.3keV microm(-1)), 244Cm alpha-particles (140-190 keV microm(-1)), 4He ions (97 keV microm(-1)), 56Fe ions (143 keV microm(-1)) or 197Au ions (1,440 keV microm(-1)) . The irradiated samples were then incubated with endo-III . SSB and DSB yields were quantified by agarose gel electrophoresis . RESULTS: Endo-III incubation produced an increase in the SSB and DSB yields . The increases were in general lower after the high LET irradiation than after gamma-irradiation . This may reflect inhibition of the activity of endo-III by the nearby DNA damage expected from high LET radiation . It can be shown that even if the activity of endo remains unchanged, significantly lower increases in SSB and DSB yields would still be expected . CONCLUSION: The results provide evidence for clustered DNA damage after high LET irradiation.

Water Res, 2001 Mar, 35(4), 1061 - 5
Effect of agitation, turbidity, aluminium foil reflectors and container volume on the inactivation efficiency of batch-process solar disinfectors; Kehoe SC et al.; We report the results of experiments designed to improve the efficacy of the solar disinfection of drinking water, inactivation process . The effects of periodic agitation, covering the rear surface of the container with aluminium foil, container volume and turbidity on the solar inactivation kinetics of Escherichia coli (starting population = 10(6) CFU ml(-1)) were investigated . It was shown that agitation promoted the release of dissolved oxygen from water with subsequent decrease in the inactivation rates of E . coli . In contrast, covering the rear surface of the solar disinfection container with aluminium foil improved the inactivation efficiency of the system . The mean decay constant for bacterial populations in foil-backed bottles was found to be a factor of 1.85 (std . dev . = 0.43) higher than that of non-foil-backed bottles . Inactivation rates decrease as turbidity increases . However, total inactivation was achievable in 300 NTU samples within 8 h exposure to strong sunshine . Inactivation kinetics was not dependent on the volume of the water container for volumes in the range 500-1500 ml.

J Perinat Med, 2001, 29(1), 36 - 41
Detection of cervical immunoglobulin A in normal pregnancy; Abitzsch S et al.; The aim of our study was to evaluate a feasible method to quantify the immunoglobulin A concentration in the cervical mucus of women with a normal singleton pregnancy . In 60 immunologic healthy pregnant women cervical mucus samples were taken at a random time in pregnancy using an absorbing cylindrical cotton-swab stick . In this cervical mucus immunoglobulin A concentration was measured by radial immunodiffusion . A vaginal bacterial swab was taken from each woman . Concentration of immunoglobulins in maternal serum was estimated . There was no statistically significant difference of cervical immunoglobulin A concentration between the investigated groups (p = 0.952): 18-24 gestational weeks (gw): 52.8 (6.6-258.4) mg/l; 25-29 gw: 89.3 (4.8-193.8) mg/l; 30-34 gw: 55 (1.4-326) mg/l; 35-40 gw: 59.2 (4-400.9) mg/l . Women with a normal vaginal flora showed a significantly higher cervical immunoglobulin A concentration than those with a pathological colonization: 92.2 (6.6-400.9) mg/l vs . 42.5 (1.4-326) mg/l (p < 0.05) . The serum levels of immunoglobulins A, A1, A2, M and G do not correlate to cervical immunoglobulin A levels nor to gestational age . In normal pregnancy, cervical immunoglobulin A concentration does not change with advancing gestational age, but a pathological vaginal colonization seems to be associated with decreased immunoglobulin A levels.

Genetika, 2001 Jan, 37(1), 46 - 53
{Cytotoxic activity of melittin expressed by recombinant vectors in Acholeplasma laidlawii and Mycoplasma hominis cells}; Aleksandrova NM et al.; Recombinant plasmids containing the mellitin gene under the control of the lac and the tetM gene promoters were used for studying cytotoxic activity of mellitin in cells of mollicutes (mycoplasmas) and Escherichia coli . After transformation of Acholeplasma laidlawii and Mycoplasma hominis cells with recombinant plasmid DNAs by electroporation, cell growth was suppressed . The expression of the mellitin gene in A . laidlawii and M . hominis cells was demonstrated using the reverse transcription polymerase chain reaction (RT-PCR) . The possibility of using the mellitin gene in the recombinant vector as a potential antimycoplasmic gene-therapeutic agent with its selective expression in target cells is discussed.

Mol Biol (Mosk), 2001 Jan-Feb, 35(1), 79 - 82
{Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301}; Rastorguev SM et al.; A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301 . In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI) . Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins . The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs . The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs . A total negative charge was assumed to contribute to the antirestriction activity.

RNA, 2001 Feb, 7(2), 302 - 13
Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation; Gilbert W et al.; Mammalian SR proteins are currently thought to function in mRNA export as well as splicing . They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides . Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear . Npl3 is a shuttling protein in budding yeast that we showed previously to be a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1 . Here we demonstrate that Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides . Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p . The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo . We propose that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export.

RNA, 2001 Feb, 7(2), 293 - 301
Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY; Jagath JR et al.; Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA . In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect . A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects . Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant . Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C . These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.

RNA, 2001 Feb, 7(2), 220 - 32
New insight into RNase P RNA structure from comparative analysis of the archaeal RNA; Harris JK et al.; A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria . The differences between the secondary structure models of archaeal and bacterial RNase P RNA have largely disappeared, and even variation in the sequence and structure of the RNAs are similar in extent and type . The structure of the cruciform (P7-11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences.

Scand J Infect Dis, 2001, 33(2), 145 - 52
Long-lasting recall response of CD4+ and CD8+ alphabeta T cells, but not gammadelta T cells, to heat shock proteins of francisella tularensis; Ericsson M et al.; Decades after recovery from tularemia, circulating alphabeta T cells are known to still recognize a variety of membrane proteins of Francisella tularensis . We studied the T cell response to 3 cytoplasmic heat shock proteins of the organism: DnaK, chaperone-60 (Cpn-60) and Cpn-10 . Determination of subpopulations of responding T cells was of special interest as it has been suggested that homologs of these conserved proteins may be recognized by human gammadelta T cells . Compared with reference subjects with no history of tularemia or tularemia vaccination, subjects who had been infected with tularemia 10-30 y earlier showed a significantly (p = 0.01) higher proliferative T cell response to all 3 heat shock proteins . In general, the magnitude of responses of CD4 T cells was higher than that of CD8 T cells . By flow cytometry, blast cells were shown to express the alphabeta T cell receptor . Under conditions that allowed vigorous expansion of gammadelta T cells in response to a phosphorylated non-peptide antigen, no expansion of gammadelta T cells occurred in response to DnaK or Cpn60 of F . tularensis . In conclusion, a long-lasting recall response to heat shock proteins of F . tularensis was demonstrated in alphabeta T cells but not in gammadelta T cells . The results support the assumption that human alphabeta T cells recognize bacterial proteins irrespective of the nature or localization of the proteins in the bacterial cell and thereby contribute to the maintenance of a long-lasting broad T cell response based on a wide variety of specificities.

Theriogenology, 2001 Jan 15, 55(2), 563 - 71
Effect of subclinical uterine infection on cervical and uterine involution, estrous activity and fertility in postpartum buffaloes; Usmani RH et al.; Nili-Ravi buffaloes (n=29) that calved normally between August and November and did not develop any clinical reproductive disorder after calving were studied for the incidence of sub-clinical bacterial infection of the uterus and its effects on postpartum reproductive efficiency . The incidence of subclinical uterine infection was 24% (7/29) . Involution of the cervix and uterus was slower (P < 0.01) in the infected group than in the normal group (45.6 vs 31.1 days and 46.3 vs 35.8 days), respectively . The mean diameters of cervix and gravid horn on Day 12 post partum and on completion of involution did not differ between buffaloes of the two groups . However, the rate of involution of the cervix and the gravid horn was lower in buffaloes of the infected group (2.2 vs . 2.7 mm/day and 2.6 vs . 3.2 mm/day) . The mean interval to first post partum ovulation was similar in buffaloes in the infected (35.5 days) and the normal group (33.8 days) . The life span of corpus luteum formed after first ovulation was shorter (11 days) in buffaloes of both groups than that of a normal estrous cycle (15 to 17 days) . The incidence of silent ovulation was apparently higher in buffaloes of the infected group (83 vs . 60%) but the difference was not significant . For the first four months after calving, the mean interval to first postpartum estrus was longer in buffaloes of the infected group (73.0 vs . 47.7 days; P < 0.01) . Similarly, the average service period was longer in buffaloes of the infected group (91.0 vs . 64.8 days; P < 0.05) . The overall pregnancy rate for the first four months after calving did not differ between buffaloes of the two groups . We conclude that subclinical bacterial infection of the postpartum uterus delays the cervical and uterine involution which can, in turn, delay the occurrence of first postpartum estrus and prolong the service period in buffaloes.

J Med Microbiol, 2001 Mar, 50(3), 233 - 7
Enhanced production of vascular endothelial growth factor by human monocytic cells stimulated with endotoxin through transcription factor SP-1; Sakuta T et al.; The effect of endotoxin on the regulation of vascular endothelial growth factor (VEGF) mRNA expression in human monocytic (THP-1) cells was examined . Endotoxic lipopolysaccharide (LPS) from Escherichia coli and synthetic E . coli-type lipid A (LA-15-PP) enhanced VEGF mRNA expression . LPS-induced VEGF mRNA accumulation was regulated, at least in part, at the transcriptional level . Enhancement of VEGF gene expression by LPS was shown by gel shift analysis and use of transcription factor inhibitors to be mediated via the activation of SP-1.

BMC Neurosci . 2001;2(1):2 . Epub 2001 Feb 16.
Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina; Horbinski C et al.; BACKGROUND: Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons . Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons . Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons . Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells . Cultures were then transfected with varying amounts of a plasmid encoding either E . coli beta-galactosidase or enhanced green fluorescence protein (EGFP) using PEI . RESULTS: Optimal transfection efficiency was observed with 1 microg/ml of plasmid DNA and 5 microg/ml PEI . Expression of beta-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days . A maximum yield was obtained within 72 hours with approximately 9% of the neurons expressing beta-galactosidase, as assessed by both histochemistry and antibody staining . Cotransfection of two plasmids encoding reporter genes was achieved . Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector . CONCLUSIONS: These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.

Nat Cell Biol, 2001 Mar, 3(3), 267 - 75
A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation; Selvarajan S et al.; Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation . Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture . Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution . PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation . Human plasma lacking PKal does not support differentiation of 3T3-L1 cells . PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis . We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.

Nat Biotechnol, 2001 Mar, 19(3), 248 - 52
Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel; Vercoutere W et al.; RNA and DNA strands produce ionic current signatures when driven through an alpha-hemolysin channel by an applied voltage . Here we combine this nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale . Measurable properties include duplex stem length, base pair mismatches, and loop length . This nanopore instrument can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.

Surgery, 2001 Mar, 129(3), 255 - 66
Expression of inducible nitric oxide synthase in the liver of bile duct-ligated Wistar rats with modulation by lymphomononuclear cells; Arriero MM et al.; BACKGROUND: The current study evaluated whether biliary tract obstruction stimulates inducible nitric oxide synthase (iNOS) protein expression in the liver and analyzed the implication of lymphomononuclear cells and interleukin-4 (IL-4) . METHODS: Male Wistar rats were used . Bile flow interruption was achieved by a complete division of the extrapancreatic common bile duct . iNOS expression was determined by both the Western blot technique and immunohistochemistry . RESULTS: iNOS protein was markedly expressed in the liver 7 days after bile duct obstruction . Treatment with thymostimulin (TP-1), a partially purified thymic extract, reduced the intensity of the expression of iNOS protein in the liver after bile duct ligation . Recent data have suggested that IL-4 attenuates iNOS protein expression . We then analyzed the involvement of this anti-inflammatory cytokine on the modulation of iNOS expression in the liver . The liver from rats that underwent bile duct ligation (BDL) showed a lower content of IL-4 than that of sham-operated (SO) rats . TP-1 treatment increased the content of IL-4 in the liver . Liver slices incubated in vitro with Escherichia coli lipopolysaccharide (LPS, 10 microg/mL) stimulated the expression of iNOS protein . The level of LPS-induced iNOS expression was reduced by lymphomononuclear cells obtained from sham-operated animals . However, lymphomononuclear cells isolated from BDL rats potentiated the induction of iNOS expression by LPS-stimulated liver . However, lymphomononuclear cells from TP-1-treated BDL rats failed to modify LPS-stimulated iNOS expression . The different effect of lymphomononuclear cells on the modulation of iNOS expression in the liver was associated with their ability to generate IL-4 . CONCLUSIONS: The liver of jaundiced rats markedly expressed iNOS protein, which was associated to modifications in the content of IL-4 in the liver . Furthermore, lymphomononuclear cells modulate iNOS protein expression in the liver by a mechanism in which IL-4 is involved.

J Invest Dermatol, 2001 Mar, 116(3), 374 - 9
Identification of cDNA encoding a serine protease homologous to human complement C1r precursor from grafted mouse skin; Byun SJ et al.; We isolated a cDNA clone from grafted mouse skin that encodes a serine protease homologous to human C1r . The C1r protease is involved in the activation of the first component of the classical pathway in the complement system . In order to identify novel transcripts whose expression is regulated in grafted mouse skin, we first performed differential display reverse transcription polymerase chain reaction analysis and obtained 18 partial cDNA clones whose protein products are likely to play an important role in allograft rejection . One of these showed significant sequence homology with human complement C1r precursor . The other clones displayed no homology to any known sequences, however . Northern blot analysis demonstrated that the level of this transcript was upregulated in day 8 postgrafted skin . The full-length cDNA 2121 nucleotides in length obtained from screening a mouse skin cDNA library contained a single open reading frame encoding 707 amino acid residues with a calculated molecular weight of 80,732 Da . Its deduced amino acid sequence revealed an 81% identity and 89% similarity to the human C1r counterpart . In particular, mouse C1r contained His501, Asp559, and Ser656, which were conserved among this group of serine proteases . This protein was thus designated as mouse C1r . We have expressed a truncated fragment of C1r protein without the N-terminal hydrophobic sequence in Escherichia coli and generated a polyclonal antibody against it . Subsequent immunohistochemical analysis confirmed that mouse C1r was significantly expressed 8 d after the skin graft in both allografted and autografted skins, compared with normal skins . These collective data suggest that a component of the complement system, C1r, might contribute to the graft versus host immune responses in mice.

Eur J Biochem, 2001 Mar, 268(5), 1477 - 85
Heat capacity analysis of oxidized Escherichia coli thioredoxin fragments (1--73, 74--108) and their noncovalent complex . Evidence for the burial of apolar surface in protein unfolded states; Georgescu RE et al.; We have calculated the absolute heat capacities of fragments 1--73 (N fragment) and 74--108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry . We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ x K(-1) x mol(-1) lower than the fully solvated-chain values . We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity) . Illustrative calculations suggest that burial of about 1000--1600 A(2) of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface) . In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.

Eur J Biochem, 2001 Mar, 268(5), 1422 - 9
Different sensitivity of rabbit heart and skeletal muscle to endotoxin-induced impairment of mitochondrial function; Trumbeckaite S et al.; The involvement of mitochondrial dysfunction in septic disturbances of tissues is controversial . The aim of this study was to investigate the effects of endotoxin-induced sepsis on the function of heart and skeletal muscle mitochondria . Rabbits were made septic by subcutaneous injection of endotoxin (lipopolysaccharide, LPS) from Escherichia coli at concentrations of 100 or 150 microg LPS.kg(-1) 24 h prior to the experiments . Mitochondrial respiration was measured in saponin-skinned muscle fibers and compared with photometrically detected activities of respiratory chain enzymes as well as with function of perfused hearts . In heart fibers a dosage of 100 microg LPS.kg(-1) caused a significant decrease of state 3-respiration for the substrates pyruvate (-38%), octanoyl-carnitine (-38%) and succinate (-30%) with correspondingly decreased respiratory control indexes (RCI) . In addition, endotoxin caused a decreased temporal stability of the rate of state 3-respiration . At least in part these changes can be attributed to a reduced activity of complex I + III (-50%) of the respiratory chain . State 4-respiration rates were not significantly altered . The lowered state 3-respiration in heart mitochondria seems to contribute to the impairment of heart muscle function as detected by an increase of coronary vascular resistance (CVR) in endotoxin-treated hearts . Functional properties of mitochondria from M . Vastus lasteralis were not affected by 100 microg LPS.kg(-1) but a higher dosage of 150 microg LPS.kg(-1) caused decreased RCI for the substrates pyruvate (-29%) and octanoyl-carnitine (-32%) . Also the activity of complex I + III was not significantly affected at lower dose of endotoxin but decreased (-42%) after treatment with 150 microg LPS.kg(-1) . Results demonstrate the involvement of impaired mitochondria in the pathophysiology of septic organ failure and a tissue specificity of endotoxaemia.

Eur J Biochem, 2001 Mar, 268(5), 1410 - 21
Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins . Purification, characterization and gene cloning; Yamaguchi S et al.; A novel protein-deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned . The enzyme is a monomer with a pI of 10.0, a measured M(r) of approximately 20,000 and a calculated M(r) of 19,860 . Extensive comparison with Streptoverticillium transglutaminase showed that the protein-deamidating enzyme lacked transglutaminase activity in terms of hydroxamate-formation between benzyloxycarbonyl-Gln-Gly and hydroxylamine, or monodansylcadaverine incorporation into casein . The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (k(cat)/K(m)) than with short peptides . The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides . The enzyme was therefore named protein-glutaminase (EC 3.5.1) . The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had protein-glutaminase activity and cross-reacted with antiserum raised against the purified enzyme . The protein-glutaminase was shown to be expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a pro-sequence of 114 amino acids . The amino-acid sequence had no obvious homology to any published sequence and is therefore a novel protein-glutaminase.

Eur J Biochem, 2001 Mar, 268(5), 1404 - 9
Thioredoxin peroxidases of the malarial parasite Plasmodium falciparum; Rahlfs S et al.; The open reading frames of two different proteins with homologies to 2-Cys peroxiredoxins have been identified in the P . falciparum genome . Both genes, with a length of 585 and 648 bp, respectively, were amplified from a gametocyte cDNA and overexpressed in Escherichia coli . The gene products (deduced m 21.8 and 24.6 kDa) with an overall identity of 51.8% were found to be active in the glutamine synthetase protector assay . The smaller protein (named Pf-thioredoxin peroxidase 1; PfTPx1) is reduced by P . falciparum thioredoxin (PfTrx) and accepts H(2)O(2), t-butylhydroperoxide, and cumene hydroperoxide as substrates, the respective k(cat) values for the N-terminally His-tagged protein in the presence of 10 microM PfTrx and 200 microM substrate being 67, 56, and 41 min(-1) at 25 degrees C . As described for many peroxiredoxins, PfTPx1 does not follow saturation kinetics . Furthermore, in oxidizing milieu both proteins are converted to another protein species migrating faster in SDS gel electrophoresis . For PfTPx1 also this second species was found to be active, however, with different kinetic properties which might indicate a mechanism of enzyme regulation in vivo.

Eur J Biochem, 2001 Mar, 268(5), 1382 - 91
Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants; De Wilde G et al.; Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells . Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression . To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli . Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known . The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution . This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template . In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations . Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD . The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.

FEBS Lett, 2001 Jan 26, 489(1), 19 - 24
Redox-regulated chaperone function and conformational changes of Escherichia coli Hsp33; Raman B et al.; We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions . While oxidized Hsp33 completely prevents the heat-induced aggregation of zeta-crystallin at 42 degrees C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation . Far UV-circular dichroism (CD) spectra show that reduced Hsp33 contains a significant alpha-helical component . Oxidation results in significant changes in the far UV-CD spectrum . Near UV-CD spectra show changes in tertiary structural packing upon oxidation . Polarity-sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33 . Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets.

Hepatology, 2001 Mar, 33(3), 591 - 6
Soluble liver antigen: isolation of a 35-kd recombinant protein (SLA-p35) specifically recognizing sera from patients with autoimmune hepatitis; Volkmann M et al.; Autoantibodies to soluble liver antigen (SLA) are considered a specific marker of autoimmune hepatitis . We have performed immunoscreening of a human liver gene expression library with an anti-SLA-positive serum . A reactive clone with a 35-kd open reading frame (ORF) and a 563 base pair (bp) 3' untranslated region (UTR) was isolated (soluble liver antigen {SLA}-p35), showing strong homology to an independently isolated putative SLA/liver-pancreas antigen (LP) sequence (Acc . No . AF146396), and a UGA serine tRNA-protein complex (tRNP)((Ser) Sec) related protein (AJ238617), as well as different expression sequence tag (EST)-clones from lymphatic and oncofetal tissues . Expressed in Escherichia coli, SLA-p35 showed dose-dependent and complete blocking of reactivity to native SLA antigen after preabsorption with the 35-kd recombinant protein . It recognized 67/85 (78.8%) precharacterized anti-SLA-positive sera in dilutions up to 1:40,000 in immunoblot, without detectable cross reactivity in the controls . The commercially available SLA/LP enzymelinked immunosorbent assay (ELISA), by comparison, recognized 63/85 samples (74.1%) . Of the negative samples, 18% showed strong inhibition rates (80% and above) in the polyclonal inhibition ELISA . We conlude that the complementary DNA now isolated by 3 independent approaches encodes for the major but not sole antigenic component of soluble liver antigen . Although its truncated form presented here may serve to improve diagnostics based on the new recombinant polypeptide, it currently cannot fully replace the polyclonal inhibition ELISA.

Plant Cell Physiol, 2001 Feb, 42(2), 107 - 13
The Arabidopsis sensor His-kinase, AHk4, can respond to cytokinins; Suzuki T et al.; His-to-Asp (His-->Asp) phosphorelay mechanisms are presumably involved in propagation of certain environmental stimuli, including phytohormones, in Arabidopsis thaliana . In addition to the previously characterized His-kinases, namely, the ETR1 family of ethylene receptors, CKI1 cytokinin-sensor, and ATHK1 osomo-sensor, this higher plant has three more His-kinases (named AHK2, AHK3, and AHK4) . By employing the well-known His-->Asp phosphorelay systems in both the fission yeast and Escherichia coli, evidence is presented showing that the AHK4 His-kinase has an ability to serve as a cytokinin-responsive environmental sensor . Taking advantage of this AHK4-dependent His-->Asp phosphorelay system in E . coli, a phosphorelay interaction between the Arabidopsis His-kinase and histidine-containing phosphotransmitters (AHPs) was also demonstrated for the first time.

J Clin Microbiol, 2001 Mar, 39(3), 971 - 6
Identification of enteropathogenic Escherichia coli in simian immunodeficiency virus-infected infant and adult rhesus macaques; Mansfield KG et al.; Enteropathogenic Escherichia coli (EPEC) was recognized as a common opportunistic pathogen of simian immunodeficiency virus-infected rhesus macaques (Macaca mulatta) with AIDS . Retrospective analysis revealed that 27 of 96 (28.1%) animals with AIDS had features of EPEC infection, and EPEC was the most frequent pathogen of the gastrointestinal tract identified morphologically . In 7.3% of animals dying with AIDS, EPEC represented the sole opportunistic agent of the gastrointestinal tract at death . In 20.8% of cases, it was seen in combination with one or more gastrointestinal pathogens, including Cryptosporidium parvum, Enterocytozoon bieneusi, Mycobacterium avium, Entamoeba histolytica, Balantidium coli, Strongyloides stercoralis, cytomegalovirus, and adenovirus . Clinically, infection was associated with persistent diarrhea and wasting and was more frequent in animals that died at under 1 year of age (P < 0.001, Fisher exact test) . The organism was associated with the characteristic attaching and effacing lesion in colonic tissue sections and produced a focal adherence pattern on a HEp-2 assay but was negative for Shiga toxin production as assessed by PCR and a HeLa cell cytotoxicity assay . A 2.6-kb fragment encompassing the intimin gene was amplified and sequenced and revealed 99.2% identity to sequences obtained from human isolates (GenBank AF116899) corresponding to the epsilon intimin subtype . Further investigations with rhesus macaques may offer opportunities to study the impact of EPEC on AIDS pathogenesis and gastrointestinal dysfunction.

Genes Dev, 2001 Feb 15, 15(4), 415 - 27
A model for the abrogation of the SOS response by an SOS protein: a negatively charged helix in DinI mimics DNA in its interaction with RecA; Voloshin ON et al.; DinI is a recently described negative regulator of the SOS response in Escherichia coli . Here we show that it physically interacts with RecA and prevents the binding of single-stranded DNA to RecA, which is required for the activation of the latter . DinI also displaces ssDNA from a stable RecA-DNA cofilament, thus eliminating the SOS signal . In addition, DinI inhibits RecA-mediated homologous DNA pairing, but has no effect on actively proceeding strand exchange . Biochemical data, together with the molecular structure, define the C-terminal alpha-helix in DinI as the active site of the protein . In an unusual example of molecular mimicry, a negatively charged surface on this alpha-helix, by imitating single-stranded DNA, interacts with the loop L2 homologous pairing region of RecA and interferes with the activation of RecA.

EMBO J, 2001 Mar 1, 20(5), 1192 - 202
Physical interactions between DinI and RecA nucleoprotein filament for the regulation of SOS mutagenesis; Yasuda T et al.; The Escherichia coli dinI gene is one of the LexA-regulated genes, which are induced upon DNA damage . Its overexpression conferred severe UV sensitivity on wild-type cells and resulted in the inhibition of LexA and UmuD processing, reactions that are normally dependent on activated RecA in a complex with single-stranded (ss)DNA . Here, we study the mechanism by which DinI inhibits the activities of RecA . While DinI neither binds to ssDNA nor prevents the formation of RecA nucleoprotein filament, it binds to active RecA filament, thereby inhibiting its coprotease activity but not the ATPase activity . Furthermore, even under in vitro conditions where UmuD cleavage dependent on RecA-ssDNA-adeno sine-5'-(3-thiotriphosphate) is blocked in the presence of DinI, LexA is cleaved normally . This result, taken together with electron microscopy observations and linear dichroism measurements, indicates that the ternary complex remains intact in the presence of DinI, and that the affinity to the RecA filament decreases in the order LexA, DinI and UmuD . DinI is thus suited to modulating UmuD processing so as to limit SOS mutagenesis.

EMBO J, 2001 Mar 1, 20(5), 1164 - 72
Mutations in DnaA protein suppress the growth arrest of acidic phospholipid-deficient Escherichia coli cells; Zheng W et al.; Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC) . Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions . Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not . The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA . Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K) . Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.

EMBO J, 2001 Mar 1, 20(5), 1042 - 50
Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone; Rudiger S et al.; The evolutionarily conserved DnaJ proteins are essential components of Hsp70 chaperone systems . The DnaJ homologue of Escherichia coli associates with chaperone substrates and mediates their ATP hydrolysis-dependent locking into the binding cavity of its Hsp70 partner, DnaK . To determine the substrate specificity of DnaJ proteins, we screened 1633 peptides derived from 14 protein sequences for binding to E.coli DnaJ . The binding motif of DnaJ consists of a hydrophobic core of approximately eight residues enriched for aromatic and large aliphatic hydrophobic residues and arginine . The hydrophobicity of this motif explains why DnaJ itself can prevent protein aggregation . Although this motif shows differences from DnaK's binding motif, DnaJ and DnaK share the majority of binding peptides . In contrast to DnaK, DnaJ binds peptides consisting of L- and D-amino acids, and therefore is not restricted by backbone contacts . These features allow DnaJ to scan hydrophobic protein surfaces and initiate the functional cycle of the DnaK system by associating with hydrophobic exposed patches and subsequent targeting of DnaK to these or to hydrophobic patches in spatial neighbourhood.

EMBO J, 2001 Mar 1, 20(5), 990 - 7
Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus; Leonard PM et al.; The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine-responsive regulatory protein is the archetype . Its crystal structure has been determined at 2.9 A resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile . The structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed alpha/beta character reminiscent of a number of RNA- and DNA-binding domains . Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel beta-sheets of the C-terminal domain, and further interactions lead to octamer formation . The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression . A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.

EMBO J, 2001 Mar 1, 20(5), 961 - 70
Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function; Sianidis G et al.; SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase . SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs . We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2 . NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery . IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controlling ADP release and optimal ATP catalysis at NBD1 . IRA2 is flexible and can undergo changes in its alpha-helical content . The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function . Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented . Two DEAD family motifs in IRA2 are essential for IRA2-NBD1 binding, optimal nucleotide turnover and polypeptide translocation . We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1 . DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.

Appl Environ Microbiol, 2001 Mar, 67(3), 1025 - 9
Protein trans-splicing to produce herbicide-resistant acetolactate synthase; Sun L et al.; Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously . In this study, we used the DnaE intein from Synechocystis sp . strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli . The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments . Cotransformation of E . coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E . coli cells . These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.

Antioxid Redox Signal, 2000 Fall, 2(3), 519 - 28
Detection of thioredoxin in gastric cancer: association with histological type; Noda N et al.; Thioredoxin (TRX) is a redox-active protein with multiple intra- and extracellular functions . This protein exists ubiquitously in all life forms, from primitive living cells, such as Escherichia coli and yeast, to higher mammals . Recently, augmentation of the expression and transcription level of TRX has been reported in tumors of various organs . In this study, we examined the expression of TRX in gastric cancer with respect to its histological type and depth of invasion . The association with cell proliferation was also studied . Results of histochemical analysis of surgical specimens as well as cytochemical analysis and Northern blot analysis of gastric cancer cell lines indicated that TRX is predominantly expressed in undifferentiated type gastric cancer rather than in the differentiated type . Neither the depth of tumor invasion nor cell proliferation significantly determined the staining intensity for TRX.

Vaccine, 2001 Feb 28, 19(15-16), 2181 - 9
Plasmids encoding granulocyte-macrophage colony-stimulating factor and CD154 enhance the immune response to genetic vaccines; Burger JA et al.; We examined whether plasmids encoding granulocyte-macrophage colony-stimulating factor (pGM-CSF) or CD40-ligand (pCD40L) could modify the immune response to antigen encoded by co-injected plasmid DNA . For this we used as antigen Escherichia coli beta galactosidase (beta-gal), encoded by the plasmid pLacZ . We found that intradermal co-injection of pLacZ with both pGM-CSF and pCD40L enhanced the anti-beta-gal IgG response by approximately two orders of magnitude compared to injections of pLacZ alone . Co-injection of both pGM-CSF and pCD40L with pLacZ significantly enhanced antigen-specific IgG, and in particular IgG(2a), over that of animals co-injected with pLacZ and either pGM-CSF or pCD40L . We found that co-injection of pGM-CSF and pCD40L with pLacZ enhanced the generation of beta-gal-specific cytotoxic T cells, and allowed for a significant expansion of CD8(+) T cells from splenocytes co-cultured with beta-gal expressing stimulator cells . The immunostimulatory effects induced by pGM-CSF or pCD40L required injection of these plasmids to the same site that received pLacZ . 'Priming' experiments, where the site of injection was pre-injected with either plasmid adjuvant, showed that pGM-CSF, but not pCD40L, could enhance the anti-beta-gal immune response induced by subsequently administered plasmid antigen . We conclude that plasmids encoding GM-CSF and CD154 are particularly effective genetic adjuvants when used together to enhance the humoral and cellular immune response to a plasmid-encoded antigen.

Vaccine, 2001 Feb 28, 19(15-16), 2071 - 9
Effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin (LT H44A) as an adjuvant for nasal influenza vaccine; Hagiwar Y et al.; The effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin, LT H44A (His to Arg substitution at position 44 from the N-terminus of the A1 fragment of the A subunit) as an adjuvant for nasal influenza vaccine were examined . (1) When 0.2 microg of LT H44A, together with 0.2 microg of influenza A/PR/8/34 virus (PR8, H1N1) vaccine, was administered intranasally into BALB/c mice (twice, 4 weeks apart), anti-PR8 hemagglutinin (HA) IgA and IgG antibody (Ab) responses were induced at levels that were sufficient to provide either complete protection against infection with a small volume of PR8 virus suspension or partial protection against infection with a lethal dose of the suspension . The dose of the mutant LT and vaccine used here (0.2 microg/ 20 g doses mouse) corresponded to the estimated dose per person, i.e . 0.1 mg/10 kg body weight . (2) Using these vaccination conditions, no additional total IgE Ab responses were induced . (3) The mutant was confirmed to be less toxic than the native LT when the toxicity was analyzed either using Y1 adrenal cells in vitro (1/483 EC(50)) or by an ileal loop test . (4) One hundred micrograms of the mutant, administered intranasally or intraperitoneally into guinea-pigs (Heartley strain, 0.3-0.4 kg), caused no body-weight changes 7 days after administration, although 100 microg of the native LT administered intraperitoneally caused death in all guinea-pigs due to diarrhea within 2 days . The intranasal administration of 100 microg of the mutant resulted in almost no pathological changes in the nasal mucosa 3 days after administration . These results suggest that LT H44A, which can be produced in high yields in an E . coli culture (about 5 mg/l), could be used as one of the effective and safe adjuvants for nasal influenza vaccine in humans.

Parasitol Int, 2000 Jan, 48(3), 215 - 22
Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli; Nakajima-Nakano K et al.; The nucleoside triphosphate hydrolase (NTPase) isoforms termed, NTPase-I and NTPase-II of Toxoplasma gondii, were expressed in Escherichia coli as inclusion bodies and purified under denaturing condition . Furthermore, NTPase-I was refolded as an active form and purified under non-denaturing condition . The purified NTPase isoforms, both denatured and refolded, were tested for their usefulness as antigens for the serodiagnosis of acute toxoplasmosis in immunocompetent humans . The test was conducted by using the recombinant NTPase isoforms and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer . Seventy-three sera from dye test-positive patients, and 30 sera from subjects with no T . gondii infection were examined . The total positive rates in dye test positive sera were: 82% (60/73) for denatured NTPase-I; 78% (57/73) for denatured NTPase-II; and 63% (46/73) for refolded NTPase-I . For all three antigen types of recombinant NTPase, the positive rates of sera of acute toxoplasmosis suspected patients were 93% (13/14) . A moderate correlation between the ELISA absorbance using these antigens and the dye test titer was observed with the correlation coefficients, 0.583 (r2) for denatured NTPase-I, 0.472 (r2) for denatured NTPase-II, and 0.604 (r2) for refolded NTPase-I in the linear regression analysis . There was no significant difference observed in the antigenicity between refolded and denatured NTPase-I, nor between the isoforms.

J Biochem (Tokyo), 2001 Mar, 129(3), 469 - 75
Effect of ions and nucleotides on the interactions of yeast Rad51 protein with single-stranded oligonucleotides; Kim JM et al.; Rad51 protein is a eukaryotic homologue of RecA protein that is essential for homologous recombination . We developed a simple procedure for purifying yeast Rad51 protein, characterized its interaction with DNA, and compared it with those of RecA from Escherichia coli and Rad51 from higher eukaryotes . Fractionation of crude extract with 0.2% polyethylenimine eliminated contaminant proteins and nucleic acids, which can perturb the subsequent purification steps . Binding of Rad51 to single-stranded DNA was detected in solution by measuring the fluorescence anisotropy of a fluorescein probe attached to the 5' end of the oligonucleotides . The interaction was stabilized by ATP, as is that of RecA, but was neither stabilized by a non-hydrolysable analog of ATP, nor destabilized by ADP, unlike the interaction of RecA . This character was very similar to that of Xenopus XRad51.1, although the binding of yeast Rad51 to DNA was more sensitive to Mg(2+) ion in both the presence and absence of ATP, and was optimal at 5--10 mM Mg(2+) . The dissociation of Rad51 protein from DNA is not, therefore, favored by the hydrolysis of ATP to ADP, in contrast to that of RecA . On the other hand, the high DNA-binding state of the Rad51-DNA complex promoted by ATP appeared to be short-lived . These features may be linked to the lower activity of Rad51 and the fact that Rad51 activity does not require the hydrolysis of ATP.

J Biochem (Tokyo), 2001 Mar, 129(3), 343 - 50
The OmpR-family of proteins: insight into the tertiary structure and functions of two-component regulator proteins; Itou H et al.; The Escherichia coli DNA-binding protein OmpR is the best characterized of those regulator proteins making up "two-component system," the simplest known form of bacterial signal transduction systems . Previous inspections of the E . coli genome DNA sequences have revealed that there are 15 proteins whose amino acid sequences show extensive similarities to that of OmpR (the OmpR-family of proteins) . The three-dimensional structures of several OmpR-family proteins have been determined . In this review, we investigated the structures and amino acid sequences of this family of proteins . The results reveal several notable conservative varieties in their tertiary structures and functions.

Virus Res, 2001 Apr, 74(1-2), 119 - 32
Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase; Wells VR et al.; The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase . Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac . Several polymerases have been analyzed by this approach allowing comparisons to be drawn . The assay included RNA synthesis by 3D(pol) on an RNA template that coded for the N-terminal region of alpha-Lac . The product of this reaction was used as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT . The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli . The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac . Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases) . Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.

J Virol Methods, 2001 Apr, 92(2), 193 - 7
Detection of prawn white spot bacilliform virus by immunoassay with recombinant antigen; Zhang X et al.; Prawn white spot bacilliform virus (WSBV) is the major pathogen of prawn disease . To develop a sensitive assay for the early detection of this virus, we generated an antibody against a WSBV-specific protein, P204 . The p204 gene was cloned from a WSBV cDNA library and expressed in Escherichia coli . The peptide (P204) encoded by p204 was purified, and its antibody raised in mice . IgG fraction of the anti-P204 serum was purified using a Sepharose column and the Fab fragment was obtained by pepsin digestion . An enzyme-linked immunosorbent assay (ELISA) using this Fab fragment was developed to detect the WSBV in prawn tissues . The experiments showed that Fab of anti-P204 antibody is highly specific for WSBV, and displays a sensitivity of 10(5) viral particles/mg prawn tissues . This is the first report using the Fab fragment prepared from a recombinant antigen to detect prawn viruses by ELISA.

J Virol Methods, 2001 Apr, 92(2), 121 - 9
A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins; Kosovsky J et al.; The expression and isolation of herpes simplex virus 1 (HSV-1) immediate early (alpha) IE63 (ICP27) and of the early (beta) thymidine kinase (Tk) polypeptides in Escherichia coli JM 109 cells transformed with the PinPoint Xa-1 (Promega) plasmid construct carrying either the HSV-1 UL54 or UL23 genes are described . The resulting biotinylated fusion protein(s) could be easily induced and were purified in appropriate amounts by means of a monomeric avidin-conjugated resin (SoftLink Soft Release Avidin Resin, Promega) provided that: (1) the exponential growth of the selected transformed cells was monitored carefully; (2) the post-induction harvest interval was properly chosen; and (3) the period for adsorption to the avidin resin suitably adjusted . The isolated protein(s), although partially digested in the case of the IE63 polypeptide, were suitable antigen(s) for immunization of various animal species . Co-purification of trace amounts of endogenous biotinylated protein(s) produced in E . coli was eliminated by shortening the duration of adsorption to the avidin resin.

FEBS Lett, 2001 Feb 23, 491(1-2), 59 - 62
Overexpression of endonuclease III protects Escherichia coli mutants defective in alkylation repair against lethal effects of methylmethanesulphonate; Eide L et al.; Endonuclease III of Escherichia coli is normally involved in the repair of oxidative DNA damage . Here, we have investigated a possible role of EndoIII in the repair of alkylation damage because of its structural similarity to the alkylation repair enzyme 3-methyladenine DNA glycosylase II . It was found that overproduction of EndoIII partially relieved the alkylation sensitivity of alkA mutant cells . Site-directed mutagenesis to make the active site of EndoIII more similar to AlkA (K120W) had an adverse effect on the complementation and the mutant protein apparently inhibited repair by competing for the substrate without base release . These results suggest that EndoIII might replace AlkA in some aspect of alkylation repair, although high expression levels are needed to produce this effect.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2905 - 10 Epub 2001 Feb 20.
One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases; Cunningham FX Jr et al.; Carotenoids in the photosynthetic membranes of plants typically contain two beta-rings (e.g., beta-carotene and zeaxanthin) or one epsilon- and one beta-ring (e.g., lutein) . Carotenoids with two epsilon-rings are uncommon . We reported earlier that the Arabidopsis thaliana lycopene epsilon-cyclase (LCYe) adds one epsilon-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene beta-cyclase (LCYb) adds two beta-rings . Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var . romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon-rings: lactucaxanthin . The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon-carotene in a heterologous Escherichia coli system . Regions of the lettuce and Arabidopsis epsilon-cyclases involved in the determination of ring number were mapped by analysis of chimeric epsilon-cyclases constructed by using an inverse PCR approach . A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one epsilon-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon-rings . An R residue in this position also yields a bi-epsilon-cyclase for both the lettuce and Arabidopsis enzymes . Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2888 - 93 Epub 2001 Feb 20.
Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli; Borgnia MJ et al.; A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms . Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF . Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed . Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ . Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria . HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification . Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly . Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes . Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2) . Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2555 - 60 Epub 2001 Feb 13.
A genomic approach to gene fusion technology; Van Dyk TK et al.; Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation . In contrast to DNA array-based methods, reporter gene technology has been underused for this application . Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions . DNA sequences from plasmid-borne, random fusions of E . coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion . The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons . Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit . Thus, the collection mirrors E . coli's transcriptional wiring diagram . This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses . Accordingly, a DNA microarray-based analysis of mitomycin C-treated E . coli indicated elevated expression of expected and unanticipated genes . Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results . The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E . coli form a single operon.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2358 - 63 Epub 2001 Feb 20.
Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles; Fernandez C et al.; The (2)H,(13)C,(15)N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa . Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30 degrees C . Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained . The (13)C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein . The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene . Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2295 - 300
TRM1, a YY1-like suppressor of rbcS-m3 expression in maize mesophyll cells; Xu T et al.; The genes rbcS and rbcL encode, respectively, the small and large subunits of the photosynthetic carbon dioxide fixation enzyme ribulose bisphosphate carboxylase/oxygenase . There is a single rbcL gene in each chloroplast chromosome; a family of rbcS genes is located in the nuclear genome . These two genes are not expressed in mesophyll cells but are in adjacent bundle-sheath cells of leaves of the C4 plant Zea mays . Two regions of the maize gene rbcS-m3 are required for suppressing expression in mesophyll cells . One region is just beyond the translation termination site in the 3' region, and the other is several hundred base pairs upstream of the transcription start site . A binding site for a protein with limited homology to the viral, yeast, and mammalian transcription repressor-activator YY1 (Yin-Yang I), has now been identified in the 3' region . A maize gene for a protein with zinc fingers homologous to those of YY1 has been isolated, characterized, and expressed in Escherichia coli . The gene is designated trm1 (transcription repressor-maize 1) . The protein TRM1 binds to the YY1-like site and, in addition, TRM1 binds to two sequence regions in the 5' region of the gene that have no homology to the YY1 site . Mutagenesis or deletion of any of these three sequences eliminates repression of rbcS-m3 reporter genes in mesophyll cells.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2244 - 9
Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis; Shibata N et al.; Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis . In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed "supernatant protein factor (SPF)," which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid {Tchen, T . T . & Bloch, K . (1957) J . Biol . Chem . 226, 921-930} . Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF . Here we report the cDNA cloning and expression of SPF from rat and human . The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as alpha-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein . Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro . SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis . SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture . Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas . Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis . These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2222 - 5
Mutated plant lectin library useful to identify different cells; Yim M et al.; The 24 nucleotides comprising the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH) cDNA were randomly mutated . The mutant lectins were expressed as glutathione-S-transferase fusion proteins in Escherichia coli and 16 clones were randomly chosen . Although all of 16 recombinant lectins reacted strongly with anti-MAH polyclonal antibody, the carbohydrate-recognition domain of each was unique . As shown by agglutination studies, each mutant MAH lectin was able to bind to erythrocytes from one or more of five animal species in very distinct patterns . Thus, novel plant lectin libraries can be used to discriminate in a highly specific manner among a variety of cell types . This technology may prove to be very useful in a number of different applications requiring a high level of specificity in cell identification.

EMBO J, 2001 Jan 15, 20(1-2), 285 - 94
The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity; Behrens S et al.; The Escherichia coli periplasmic peptidyl-prolyl isomerase (PPIase) SurA is involved in the maturation of outer membrane porins . SurA consists of a substantial N-terminal region, two iterative parvulin-like domains and a C-terminal tail . Here we show that a variant of SurA lacking both parvulin-like domains exhibits a PPIase-independent chaperone-like activity in vitro and almost completely complements the in vivo function of intact SurA . SurA interacts preferentially (>50-fold) with in vitro synthesized porins over other similarly sized proteins, leading us to suggest that the chaperone-like function of SurA preferentially facilitates maturation of outer membrane proteins.

Fresenius J Anal Chem, 2001 Jan 2, 369(2), 145 - 52
Quinoprotein glucose dehydrogenase modified thick-film electrodes for the amperometric detection of phenolic compounds in flow injection analysis; Rose A et al.; The use of thick-film electrodes as basic transducers for highly sensitive amperometric biosensors using PQQ (pyrroloquinoline quinone) dependent glucose dehydrogenase (GDH) with short response times is described . The enzyme is embedded in a polyurethane matrix on top of a platinum based thick film electrode and its ability to reduce oxidized phenolic compounds is exploited . The electrochemical amplification is based on the oxidation of the analyte on the surface of the electrode followed by its enzymatic reduction . Different parameters of the glucose dehydrogenase electrode system using dopamine as a model analyte were optimized, e.g., membrane thickness, pH value, buffer system, flow rate and storage conditions . Using optimized parameters the sensitivity and detection limits for various phenolic compounds were evaluated . The comparison of electrodes from the identical as well as from different batches shows the ability to produce a number of well reproducible sensors showing remarkably small differences with respect to parameters as sensitivity, response times and measuring range.

Inorg Chem, 2001 Feb 12, 40(4), 726 - 39
Manganese(III) biliverdin IX dimethyl ester: a powerful catalytic scavenger of superoxide employing the Mn(III)/Mn(IV) redox couple; Spasojevic I et al.; A manganese(III) complex of biliverdin IX dimethyl ester, (MnIIIBVDME)2, was prepared and characterized by elemental analysis, UV/vis spectroscopy, cyclic voltammetry, chronocoulometry, electrospray mass spectrometry, freezing-point depression, magnetic susceptibility, and catalytic dismuting of superoxide anion (O2.-) . In a dimeric conformation each trivalent manganese is bound to four pyrrolic nitrogens of one biliverdin dimethyl ester molecule and to the enolic oxygen of another molecule . This type of coordination stabilizes the +4 metal oxidation state, whereby the +3/+4 redox cycling of the manganese in aqueous medium was found to be at E1/2 = +0.45 V vs NHE . This potential allows the Mn(III)/Mn(IV) couple to efficiently catalyze the dismutation of O2.- with the catalytic rate constant of kcat = 5.0 x 10(7) M-1 s-1 (concentration calculated per manganese) obtained by cytochrome c assay at pH 7.8 and 25 degrees C . The fifth coordination site of the manganese is occupied by an enolic oxygen, which precludes binding of NO., thus enhancing the specificity of the metal center toward O2.- . For the same reason the (MnIIIBVDME)2 is resistant to attack by H2O2 . The compound also proved to be an efficient SOD mimic in vivo, facilitating the aerobic growth of SOD-deficient Escherichia coli.

Shock, 2001 Feb, 15(2), 157 - 62
Effects of Ringer-acetate and Ringer-dextran solutions on the microcirculation after LPS challenge: observations in the hamster cheek pouch; de Carvalho H et al.; The effects of NaCl 0.9%, Ringer-acetate, and Ringer-dextran given as intravenous infusions in the microcirculatory changes observed in early stages of endotoxemia were investigated in male hamsters treated with Escherichia coli lipopolysaccharide (LPS) . The cheek pouch was studied in vivo by means of intravital microscopy . Mean arterial (MAP) and venous pressures (CVP), heart rate, mean arteriolar internal diameter, spontaneous arteriolar vasomotion (AV), red blood cell velocity (RBCV) in these vessels, and long-term effects of LPS were evaluated in animals treated with either LPS alone or the combination of LPS with NaCl 0.9%, Ringer-acetate and Ringer-dextran . The intravenous injection of LPS (0.3 mg/kg) elicited a significant reduction in MAP and CVP, cessation of AV and a decrease in RBCV . In our study, the heart rate and the arteriolar diameter did not change significantly, compared with the control values obtained before the LPS injection . No improvement in the MAP could be detected with infusions of NaCl 0.9% or Ringer-acetate but the infusion of Ringer-dextran increased it significantly . All infusions tested maintained the CVP until the end of the observation period and the Ringer-dextran increased it significantly . The heart rate was maintained around 360 beats/min with a tendency to decrease 70 min after the LPS infusion in all groups studied except the group which received NaCl 0.9% where the heart rate decreased significantly . In all the four groups, the mean arteriolar diameter did not change significantly with time during the observed period . RBCV decrease with the combination LPS + NaCl 0.9% and the infusions of Ringer-acetate and Ringer-dextran maintained it until the end of the observation period . The combination of LPS + NaCl 0.9% maintained the spontaneous arteriolar vasomotion during 50 min after LPS injection and the infusion of Ringer-acetate maintained it for the 3-h observation period . The infusion of Ringer-dextran maintained the amplitude of the spontaneous arteriolar vasomotion and increased its frequency significantly . The long-term effects of LPS showed weight loss and pus on the periorbital area . Our results suggest that the best solution to maintain the microcirculatory parameters during the early stage of endotoxemia after LPS injection was the Ringer-dextran.

Nat Struct Biol, 2001 Mar, 8(3), 254 - 7
GrpE accelerates peptide binding and release from the high affinity state of DnaK; Mally A et al.; The Escherichia coli nucleotide exchange factor GrpE accelerates the rate of ADP dissociation from high affinity ADP-DnaK, thus enabling ATP binding and transition to the low affinity state . We show here that GrpE, in the absence of ATP, accelerates the rates of the forward and reverse reaction ADP-DnaK-P right harpoon over left harpoon ADP-DnaK + P, where P denotes peptide substrate . Specifically, the binding of GrpE to an ADP-DnaK-P (or DnaK-P) complex increases koff and kon by approximately 200-fold and approximately 60-fold, respectively . The results are consistent with a GrpE- induced conformational change in the C-terminal polypeptide binding domain of an ADP-DnaK molecule, which results in a unique low affinity intermediate from which peptide can dissociate . A simulation of peptide dissociation from DnaK as a function of the {ATP} / {ADP} ratio shows that GrpE induced peptide dissociation from ADP-DnaK is important at elevated cellular concentrations of ADP, which typically occur upon stress.

Nat Struct Biol, 2001 Mar, 8(3), 230 - 3
Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase; Kim YI et al.; The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals . Many family members also interact with peptidases to form ATP-dependent proteases . In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase . Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E . coli ClpX that is essential for ClpP recognition . Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities . Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner . Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.

Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 469 - 71
Crystallization and secondary-structure determination of a protein of the Lrp/AsnC family from a hyperthermophilic archaeon; Kudo N et al.; A protein belonging to the Lrp/AsnC transcription-factor family, pot1216151, from the hyperthermophilic archaeon Pyrococcus sp . OT3 was crystallized . In Escherichia coli, leucine-responsive protein (Lrp) and AsnC regulate a number of metabolic genes . The crystals of pot1216151 diffracted to 2.3 A using a conventional X-ray source and to 1.8 A using a synchrotron-radiation source . The space group was identified to be P3(1)21 or P3(2)21, with unit-cell parameters a = b = 96.9, c = 98.5 A . In combination with diffraction data obtained from K(2){Pt(CN)(6)} and K(AuCl(4)) derivatives, an electron-density map was calculated at a resolution of 3.0 A . Four monomers were identified in the asymmetric unit, with four beta-strands and two alpha-helices in each monomer.

Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 454 - 6
Purification, crystallization and preliminary X-ray diffraction analysis of the fructose-1,6-/sedoheptulose-1,7-bisphosphatase of Synechococcus PCC 7942; Nakamura Y et al.; Fructose-1,6-/sedoheptulose-1,7-bisphosphatase of Synechococcus PCC 7942, overexpressed from Escherichia coli, has been purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant . The crystals were monoclinic, with unit-cell parameters a = 80.1, b = 84.2, c = 104.3 A, beta = 101.7 degrees . They belonged to space group P2(1) and diffracted to at least 2.2 A resolution . The calculated V(M) value, based on a tetramer in the asymmetric unit, was 2.2 A(3) Da(-1).

Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 448 - 50
Purification, crystallization and preliminary crystallographic analysis of the periplasmic binding protein ProX from Escherichia coli; Breed J et al.; A periplasmic binding protein (ProX) for the compatible solutes glycine betaine and proline betaine from Escherichia coli was crystallized using the hanging-drop vapour-diffusion method . Crystals were grown using a protein concentration of 10 mg ml(-1) and a precipitant of 26-28% PEG 4000 in 50 mM PIPES pH 6.2-6.4 . Native diffraction data to 1.93 A resolution have been obtained from crystals at 290 K . The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.1, b = 55.0, c = 115.7 A, and contain one molecule per asymmetric unit.

Gene, 2001 Jan 24, 263(1-2), 273 - 84
Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli; Stenstrom CM et al.; The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli . We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence . Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained . The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability . However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA . High adenine content of the +2 codon was associated with high gene expression . Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon . Analysis of the 3540 E . coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes . Codons that are associated with low gene expression are under-represented . The data suggest that evolution has favored codons at the +2 position that give high translation initiation.

Gene, 2001 Jan 24, 263(1-2), 179 - 87
Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated; Reddy MK et al.; We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea . The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region . The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively . The pea 33RNP was expressed in Escherichia coli and purified to homogeneity . The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast . The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA . The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated . Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns . We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Gene, 2001 Jan 24, 263(1-2), 151 - 8
Differential splicing of Pneumocystis carinii f . sp . carinii inosine 5'-monophosphate dehydrogenase pre-mRNA; Ye D et al.; Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms . Pneumocystis carinii f . sp . carinii IMPDH mRNA (GeneBank Accession No: U42442) previously identified from cultured organisms yielded a predicted amino acid sequence about 70 amino acids shorter at the amino terminus than IMPDH from other species . Recent research has shown that the amino terminal region is important for enzyme activity, suggesting that the previous putative P . carinii IMPDH might not represent full length, functional enzyme . To test this hypothesis, RT-PCR was performed with total RNA isolated from P . carinii f . sp . carinii . Three IMPDH splicing variants were found and splicing preference was observed: P . carinii isolated from infected rat lung contained primarily splicing variant one (introns two and four deleted), but organisms from spinner flask culture contained primarily splicing variant three (all four introns deleted) . Importantly, splicing variant one (GeneBank Accession No: AF196975) contained an open reading frame for 529 amino acids, a size comparable to that of other eukaryotic IMPDH forms . The other variants contained the same open reading frame (454 amino acids) previously reported . Sequence analysis and complementation studies suggest variant one represents the full length, catalytically active form of P . carinii IMPDH . The differential splicing of the enzyme may reflect a mechanism by which the organism regulates the expression of IMPDH in response to environmental stresses.

Gene, 2001 Jan 24, 263(1-2), 103 - 12
Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans; Timmons L et al.; Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans . Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA . This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell . Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments . When fed to C . elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants . We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males . Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.

Toxicology, 2001 Feb 28, 159(3), 135 - 41
Cytotoxicity of derivatives from dehydrocrotonin on V79 cells and Escherichia coli; da Silva Melo P et al.; New derivatives from dehydrocrotonin (DHC, compound I), with the same anti-ulcerogenic properties but less toxicity were synthesised by reducing the cyclohexenone moiety of DHC with NaBH(4) (compound II), by reducing the cyclohexenone and lactone moieties with LiAlH(4) (compound III) and by transforming the lactone moiety into an amide (compound IV) using dimethylamine . The cytotoxicity of these derivatives from DHC was assayed on V79 fibroblast cell line . Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU) . IC(50) values of 540 and 350 microM were obtained for compound II in the NRU and NAC tests, respectively . Compound III was less toxic than the other DHC derivatives (IC(50)=1800 microM) on V79 cells based on NAC assay . Compound IV showed an IC(50) ranging from 350 to 600 microM based on the three endpoints evaluated . The three compounds were less toxic on V79 cells than DHC . DHC, compounds II, III and IV did not change the respiration rate of Escherichia coli on the acute toxicity assay.

J Biotechnol, 2001 Mar 9, 86(1), 19 - 30
Development of an Escherichia coli whole cell biocatalyst for the production of L-amino acids; Wilms B et al.; A whole cell biocatalyst for the enzymatic production of L-amino acids from hydantoins was created by coexpressing the genes encoding the L-hydantoinase, the L-N-carbamoylase and the hydantoin racemase from Arthrobacter aurescens in Escherichia coli . In order to construct a well balanced reaction system the enzymatic activity in the cells was varied by using vectors with different copy numbers for expression of the genes . Derivatives of pSC101, pACYC184 and pBR322 were employed for the various constructions and in one construct the hydantoinase gene was integrated into the E . coli chromosome . All constructs carried the E . coli rhamnose promoter system enabling gene expression control by transcriptional regulation . The productivity for L-tryptophan from the corresponding hydantoin was more than 6-fold higher than achieved with Arthrobacter aurescens.

Mol Biochem Parasitol, 2001 Feb, 112(2), 219 - 28
The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system; Krnajski Z et al.; The thioredoxin system consists of the NADPH dependent disulphide oxidoreductase thioredoxin reductase (TrxR) which catalyses the reduction of the small protein thioredoxin . This system is involved in a variety of biological reactions including the reduction of deoxyribonucleotides, transcription factors and hydrogen peroxide . In recent years the TrxR of the malaria parasite Plasmodium falciparum was isolated and characterised using model substrates like 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and Escherichia coli thioredoxin . Here we report on the isolation of a cDNA encoding for P . falciparum thioredoxin (PfTrx) and the expression and characterisation of the recombinant protein, the natural substrate of PfTrxR . The deduced amino acid sequence of PfTrx encodes for a polypeptide of 11715 Da and possesses the typical thioredoxin active site motif CysGlyProCys . Both cysteine residues are essential for catalytic activity of the protein, as shown by mutational analyses . Steady state kinetic analyses with PfTrxR and PfTrx in several coupled assay systems resulted in K(m)-values for PfTrx in the range of 0.8--2.1 microM which is about 250-fold lower than for the model substrate E . coli thioredoxin . Since the turnover of both substrates is similar, the catalytic efficiency of PfTrxR to reduce the isolated PfTrx is at least 250-fold higher than to reduce E . coli thioredoxin . PfTrx contains a cysteine residue in position 43 in addition to the active-site cysteine residues, which is partially responsible for dimer formation of the protein as demonstrated by changing this amino acid into an alanine residue . Using DTNB we showed that all three cysteine residues present in PfTrx are accessible to modification by this compound . Surprisingly the first cysteine residue of the active site motif (Cys30) is less accessible than the second cysteine (Cys33), which is highly prone to the modification . These results suggest a difference in the structure and reaction mechanism of PfTrx compared to other known thioredoxins.

J Immunol Methods, 2001 Feb 1, 248(1-2), 7 - 15
Bispecific human IgG by design; Carter P; A major obstacle facing the development of bispecific antibodies as therapeutics has been the formidable task of producing these complex molecules in sufficient quantity and purity for clinical trials . These production difficulties have been largely overcome with the advent of efficient methods for the secretion of designer bispecific antibody fragments such as diabodies and miniantibodies from Escherichia coli . In contrast, the creation of bispecific IgG by the coexpression of two different IgG is highly inefficient due to unwanted pairings of the component heavy and light chains . A robust technology for the creation of bispecific IgG has recently been developed that virtually precludes IgG contaminants, as reviewed here . This technology is anticipated to spur the clinical development of bispecific IgG and other bifunctional Fc-containing molecules such as antibody/immunoadhesin hybrids and bispecific immunoadhesins.

Toxicol Sci, 2001 Mar, 60(1), 144 - 51
Development of pulmonary tolerance in mice exposed to zinc oxide fumes; Wesselkamper SC et al.; As a result of repeated exposures to inhaled toxicants such as zinc oxide (ZnO), numerous individuals acquire tolerance to the exposures and display reduced symptoms . To ascertain whether tolerance is developed in an animal model, NIH-Swiss mice were exposed to 1.0 mg/m(3) ZnO for 1, 3, or 5 days (1X, 3X, or 5X), and polymorphonuclear leukocyte (PMN) and protein levels in bronchoalveolar lavage (BAL) were measured . Mice acquired tolerance to neutrophil infiltration into the lungs, as total PMNs returned near baseline in 5X-exposed animals as compared to that of the 1X exposure group (1X = 2.7 +/- 0.4 x 10(4), 5X = 0.2 +/- 0.1 x 10(4), mean +/- SE, p < 0.05) . Development of tolerance to changes in lavageable protein, however, was not observed (1X = 313 +/- 29 microg/ml, 5X = 684 +/- 71 microg/ml, p < 0.05) . Tolerance to PMN influx did not persist following re-exposure to ZnO after 5 days of rest . In contrast to ZnO exposure, following single and repeated exposure to aerosolized endotoxin there was development of tolerance to protein in BAL (1X = 174 +/- 71 microg/ml, 5X = 166 +/- 14 microg/ml, p > 0.05), but not to PMN influx (1X = 5.5 +/- 1.7 x 10(4), 13.9 +/- 1.7 x 10(4), p < 0.05) . Induction of lung metallothionein (MT) was also observed in mice exposed once or repeatedly exposed to ZnO, suggesting that MT may play a role in its molecular mechanism.

Science, 2001 Feb 23, 291(5508), 1553 - 7
Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors; Sondermann H et al.; Bag (Bcl2-associated athanogene) domains occur in a class of cofactors of the eukaryotic chaperone 70-kilodalton heat shock protein (Hsp70) family . Binding of the Bag domain to the Hsp70 adenosine triphosphatase (ATPase) domain promotes adenosine 5'-triphosphate-dependent release of substrate from Hsp70 in vitro . In a 1.9 angstrom crystal structure of a complex with the ATPase of the 70-kilodalton heat shock cognate protein (Hsc70), the Bag domain forms a three-helix bundle, inducing a conformational switch in the ATPase that is incompatible with nucleotide binding . The same switch is observed in the bacterial Hsp70 homolog DnaK upon binding of the structurally unrelated nucleotide exchange factor GrpE . Thus, functional convergence has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide exchange.

Nucleic Acids Res . 2001 Mar 1;29(5):E27.
Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening; Giovannoni L et al.; We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration . Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse . Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies . We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis . From an antibody library of 7 x 10(8) clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence . The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised . Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose . ME4C binds to the ED-B domain with a dissociation constant K:(d) = 1 x 10(-7) M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

Nucleic Acids Res, 2001 Mar 1, 29(5), 1163 - 74
In vitro roles of invariant helix-turn-helix motif residue R383 in sigma(54) (sigma(N)); Wigneshweraraj SR et al.; In vitro DNA-binding and transcription properties of sigma(54) proteins with the invariant Arg383 in the putative helix-turn-helix motif of the DNA-binding domain substituted by lysine or alanine are described . We show that R383 contributes to maintaining stable holoenzyme-promoter complexes in which limited DNA opening downstream of the -12 GC element has occurred . Unlike wild-type sigma(54), holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC . Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the -1 site . Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element . Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex . This indicates that R383 is not part of the regulatory centre in the sigma(54) holoenzyme, which includes the -12 promoter region elements . R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of sigma(54).

Nucleic Acids Res, 2001 Mar 1, 29(5), 1114 - 24
PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping; Beaulieu M et al.; Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms . Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses . We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA . Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration . Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model . Thus, rapid, sequence-independent optimization was possible for each new genomic target region . Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated . We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes . Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

J Virol, 2001 Mar, 75(6), 2818 - 24
Sequences at the 3' untranslated region of bamboo mosaic potexvirus RNA interact with the viral RNA-dependent RNA polymerase; Huang CY et al.; The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure . These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P . Cheng and C.-H . Tsai, J . Mol . Biol . 288:555-565, 1999) . Electrophoretic mobility shift and competition assays have now been used to demonstrate that the Escherichia coli-expressed RNA-dependent RNA polymerase domain (Delta 893) derived from BaMV open reading frame 1 could specifically bind to the 3' UTR of BaMV RNA . No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3' UTR was a less efficient competitor . Competition analysis with different regions of the BaMV 3' UTR showed that Delta 893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail . Footprinting analysis revealed that Delta 893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.

J Virol, 2001 Mar, 75(6), 2753 - 64
Characterization of Rous sarcoma virus Gag particles assembled in vitro; Yu F et al.; Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA . We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM) . RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly . The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8% . The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV . By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein . In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles . Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly . Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly . Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

J Bacteriol, 2001 Mar, 183(6), 2145 - 7
Genes involved in copper homeostasis in Escherichia coli; Grass G et al.; Recently, genes for two copper-responsive regulatory systems were identified in the Escherichia coli chromosome . In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in E . coli.

J Bacteriol, 2001 Mar, 183(6), 2132 - 6
Identification of specific residues in colicin E1 involved in immunity protein recognition; Lindeberg M et al.; The basis of specificity between pore-forming colicins and immunity proteins was explored by interchanging residues between colicins E1 (ColE1) and 10 (Col10) and testing for altered recognition by their respective immunity proteins, Imm and Cti . A total of 34 divergent residues in the pore-forming domain of ColE1 between residues 419 and 501, a region previously shown to contain the specificity determinants for Imm, were mutagenized to the corresponding Col10 sequences . The residue changes most effective in converting ColE1 to the Col10 phenotype are residue 448 at the N terminus of helix VI and residues 470, 472, and 474 at the C terminus of helix VII . Mutagenesis of helix VI residues 416 to 419 in Col10 to the corresponding ColE1 sequence resulted in increased recognition by Imm and loss of recognition by Cti.

J Bacteriol, 2001 Mar, 183(6), 2059 - 70
Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli; Christoffersen CA et al.; The overlapping and opposing promoter elements for the Escherichia coli fepDGC operon and the ybdA gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector . Primary promoter structures for the opposing transcripts overlapped extensively such that their -10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart . Relative to the E . coli consensus sequence, both promoters were poorly conserved at the -35 position and mutations which strengthened the -35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression . Both the fepD and ybdA primary promoters were shown to require a 5'-TGn-3' upstream extension of their -10 elements for optimal activities . Secondary promoters were identified for both fepD and ybdA, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs . The data provided strong evidence that the architecture of the regulatory elements within the overlapping fepD and ybdA promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors . Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.

J Bacteriol, 2001 Mar, 183(6), 2046 - 50
Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF; Hazan R et al.; "Addiction modules" consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin . The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells) . The Escherichia coli chromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death . Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E . coli mazEF system . In addition, we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E . coli is inhibited . Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEF systems.

J Bacteriol, 2001 Mar, 183(6), 1997 - 2005
Hfq is necessary for regulation by the untranslated RNA DsrA; Sledjeski DD et al.; DsrA is an 85-nucleotide, untranslated RNA that has multiple regulatory activities at 30 degrees C . These activities include the translational regulation of RpoS and H-NS, global transcriptional regulators in Escherichia coli . Hfq is an E . coli protein necessary for the in vitro and in vivo replication of the RNA phage Qbeta . Hfq also plays a role in the degradation of numerous RNA transcripts . Here we show that an hfq mutant strain is defective for DsrA-mediated regulation of both rpoS and hns . The defect in rpoS expression can be partially overcome by overexpression of DsrA . Hfq does not regulate the transcription of DsrA, and DsrA does not alter the accumulation of Hfq . However, in an hfq mutant, chromosome-expressed DsrA was unstable (half-life of 1 min) and truncated at the 3' end . When expressed from a multicopy plasmid, DsrA was stable in both wild-type and hfq mutant strains, but it had only partial activity in the hfq mutant strain . Purified Hfq binds DsrA in vitro . These results suggest that Hfq acts as a protein cofactor for the regulatory activities of DsrA by either altering the structure of DsrA or forming an active RNA-protein complex.

Invest Ophthalmol Vis Sci, 2001 Mar, 42(3), 743 - 51
Human lens thioltransferase: cloning, purification, and function; Qiao F et al.; PURPOSE . To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT) . METHODS . The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay . TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis . The activity was assayed with a synthetic substrate hydroxyethyl disulfide . Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2 . RESULTS . The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues . The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells . The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase . It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources . It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro . Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells . CONCLUSIONS . The human lens TTase gene has been cloned for the first time . Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

Br J Ophthalmol, 2001 Mar, 85(3), 277 - 80
Persistence of acanthamoeba antigen following acanthamoeba keratitis; Yang YF et al.; AIM: To investigate the hypothesis that persistent corneal and scleral inflammation following acanthamoeba keratitis is not always caused by active amoebic infection but can be due to persisting acanthamoebic antigens METHODS: 24 lamellar corneal biopsy and penetrating keratoplasty specimens were obtained from 14 consecutive patients at various stages of their disease and divided for microscopy and culture . Histological sections were immunostained and screened for the presence of Acanthamoeba cysts by light microscopy . Cultures were carried out using partly homogenised tissues on non-nutrient agar seeded with E coli . Clinical data were obtained retrospectively from the case notes of these patients . RESULTS: Of the 24 specimens, 20 were obtained from eyes that were clinically inflamed at the time of surgery . Acanthamoeba cysts were present in 16 (80%) of these 20 specimens, while only five (25%) were culture positive . Acanthamoeba cysts were found to persist for up to 31 months after antiamoebic treatment . CONCLUSION: These findings support the hypothesis that Acanthamoeba cysts can remain in corneal tissue for an extended period of time following acanthamoeba keratitis and may cause persistent corneal and scleral inflammation in the absence of active amoebic infection . In view of these findings, prolonged intensive antiamoebic therapy may be inappropriate when the inflammation is due to retained antigen rather than to viable organisms

Biophys J, 2001 Mar, 80(3), 1498 - 506
Time-resolved study of the inner space of lactose permease; Nachliel E et al.; Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state . Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment . At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 x 10(5) M(-1) . The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site . The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model . The analysis indicated that the binding domain is a cleft (between 9 and 17 A deep) characterized by low activity of water (a((water)) = 0.71), reduced diffusivity of protons, and enhanced electrostatic potential . The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated . These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity . The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein . In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing.

Virology, 2001 Mar 1, 281(1), 102 - 8
In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein; Baudin F et al.; Spontaneous proteolysis of influenza virus M1 protein during crystallisation has defined an N-terminal domain of amino acids 1--164 . Full-length M1, the N-terminal domain, and the C-terminal part of M1 (residues 165--252) were produced in Escherichia coli . In vitro tests showed that only full-length M1 and its N-terminal domain bind to negatively charged liposomes and that only full-length M1 and its C-terminal part bind to RNP . However, only full-length M1 had transcription inhibition activity . Several independent experimental approaches indicate that in vitro transcription inhibition occurs through polymerisation/aggregation of M1 onto RNP, or of M1 onto M1 already bound to RNP, rather than by binding to a specific active site on the nucleoprotein or the polymerase . The structure/function of influenza virus M1 will be compared with that of the Ebola virus matrix protein, VP40 .

Cryobiology, 2000 Dec, 41(4), 319 - 23
The lower hydrolysis of ATP by the stress protein GroEL is a major factor responsible for the diminished chaperonin activity at low temperature; Mendoza JA et al.; The chaperonins GroEL and GroES were shown to facilitate the refolding of urea-unfolded rhodanese in an ATP-dependent process at 25 or 37 degrees C . A diminished chaperonin activity was observed at 10 degrees C, however . At low temperature, GroEL retains its ability to form a complex with urea-unfolded rhodanese or with GroES . GroEL is also able to bind ATP at 10 degrees C . Interestingly, the ATPase activity of GroEL was highly decreased at low temperatures . Hydrolysis of ATP by GroEL was 60% less at 10 degrees C than at 25 degrees C . We conclude that the reduced hydrolysis of ATP by GroEL is a major but perhaps not the only factor responsible for the diminished chaperonin activity at 10 degrees C . GroEL may function primarily at higher temperatures in which the ability of GroEL to hydrolyze ATP is not compromised .

Carcinogenesis, 1980, 1(10), 837 - 48
Cleavage of lambda repressor and induction of recA protein synthesis elicited by aflatoxin B1 metabolites in Escherichia coli; Moreau PL et al.; In Escherichia coli lambda lysogens incubated with activated aflatoxin B1, the rate of synthesis of RecA protein is markedly increased while lambda repressor is cleaved, but not that of the non-inducible mutant lambdacIind- . Following a 10 min lag period, lambda repressor is almost totally cleaved within 40 min of incubation . Cleavage of lambda repressor is inhibited by chloramphenicol . Synthesis of RecA protein and cleavage of lambda repressor are two characteristic processes induced in E . coil by DNA damaging agents such as u.v . light or mitomycin C . Our results favour the idea that in aflatoxin B1-treated lysogens, DNA lesions activate RecA protein to cleave LexA protein, the repressor of the recA gene, and lambda repressor . DNA damage, which may represent a relatively small fraction of the total cellular lesions, promotes the derepression of specific genes that control epigenetic as well as genetic processes in bacteria.

J Anim Sci, 2001 Feb, 79(2), 413 - 9
Dietary phosphorus and an inflammatory challenge affect performance and immune function of weanling pigs; Kegley EB et al.; Ninety-six 3-wk-old pigs (6.3+/-0.12 kg initial BW) were allotted to one of eight treatments based on BW and litter origin to determine the effect of dietary phosphorus and an inflammatory challenge on performance and immune function . Four corn-soybean meal-based treatment diets were formulated to contain 0.16, 0.24, 0.32, or 0.40% available P . Monocalcium-dicalcium phosphate was used as the supplemental P source . The Ca:available P ratio was maintained at 2:1 . To challenge the pigs, half of the pigs in each dietary treatment were injected i.m . with E . coli lipopolysaccharide (200 microg/kg of BW) on d 7 and 14 . This resulted in a 2 x 4 factorial arrangement of treatments . Average daily gain for the 35-d study was increased linearly (P < 0.01) by increasing supplemental P . Average daily gain and ADFI were decreased (P < 0.05) by lipopolysaccharide injection . Serum P concentrations increased linearly (P < 0.01) with increasing supplemental P . Antibody titers to the injection of sheep red blood cells and ovalbumin on d 21 decreased linearly (P < 0.10) by increasing supplemental P . In vitro blastogenic response of lymphocytes to phytohemagglutinin (PHA) on d 25 was increased linearly (P < 0.05) by increasing supplemental P . Blastogenic response of lymphocytes to pokeweed mitogen on d 25 was not affected . On d 31, skinfold thickness 6 h following an intradermal injection of PHA was increased quadratically (P < 0.07) by increasing supplemental P . There were no P x lipopolysaccharide interactions for any immune response measure . In conclusion, increasing supplemental P increased ADG and enhanced cell-mediated immune response but decreased humoral immune response.

Arthritis Res, 2000, 2(1), 59 - 64
Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction; Pap T et al.; AIMS: PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin . Mutations of PTEN have been described in several human cancers and associated their invasiveness and metastatic properties . Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth . In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF . In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA . Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals . Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far . The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing . For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription . For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared . To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice . After 60 days, mice were sacrificed, the implants removed and embedded into paraffin . RESULTS: PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig.1) . No additional bands that could account for mutant PTEN variants were detected . Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051) . However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes . As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA . In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b) . Normal synovial specimens showed homogeneous staining fo PTEN within the thin synovial membrane (Fig . 2c) . In situ hybridization using the sense probe gave no specific staining (Fig . 2d) . We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage . Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig . 3A), and the proportion of PTEN expressing cells did not change throughout the passages . In contrast, control experiments using a specific RNA probe fo MMP-2 revealed mRNA expression by nearly all cultured cells (Fig . 3B) . As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation . Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig . 3c) . In situ hybridization for MMP-2 showed abundant staining in these cells (Fig . 3d) . DISCUSSION: Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more that half of cultured RA-SF is of interest . It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively . It has been proposed that the tyrosine phosphatase activity of counteracting th actions o protein tyrosine kinases . As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease . However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling . The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-beta . Therefore, it could be hypothesized that TGF-beta might be responsible for the downregulation of PTEN . (ABSTRACT TRUNCATED)

Analyst, 2000 Dec, 125(12), 2274 - 9
Disposable potentiometric enzyme sensor for direct determination of organophosphorus insecticides; Gaberlein S et al.; A potentiometric disposable enzyme sensor for the direct and fast determination of organophosphorus (OP) insecticides was developed by using an organophosphorus hydrolase (OPH) immobilized on an ion-selective electrode . The disposable screen-printed transducer was based on double matrix membrane technology which allows easy mass production . The potentiometric device consisted of a H(+)-sensitive electrode with integrated Ag/AgCl reference electrode . The electrodes were prepared with N,N-dioctadecylmethylamine as H(+)-sensitive ionophore and pH calibration resulted in slopes of 55 mV decade-1 over a pH range from 11 to 6 . OPH was isolated from recombinant Escherichia coli DH5 alpha and immobilized within poly(carbamoyl sulfonate) prepolymer on the surface of the H(+)-sensitive electrode without any further fixation membrane . OPH catalyzes the hydrolytic cleavage of OP compounds which releases protons in a concentration proportional to hydrolyzed substrate . Sensor performance was investigated with regard to enzyme load, concentration, pH and temperature of the measuring buffer using paraoxon as analyte . Best sensitivity and response time were obtained with sensors prepared with 250 U of OPH and measuring at 37 degrees C in 1.0 mM HEPES buffer, pH 9.3, containing 100 mM NaCl . The enzyme sensor exhibited a linear calibration range of 0.01-0.15 mM chlorpyrifos, 0.05-0.35 mM diazinon, 0.05-0.4 mM paraoxon and 0.007-0.05 mM parathion, respectively . For all these analytes response times to reach 95% of maximum change in potential did not exceed 5 min . Sensors stored under dry conditions at 4 degrees C still showed 60% of initial hydrolytic rate after 70 d . The sensors even when stored dry were ready for measurements after 5 min incubation in measuring buffer . A range of putative interfering substances did not influence sensor response, and suitability of measuring OPs in soil extracts was ascertained.

Nature, 2001 Feb 8, 409(6821), 720 - 4
The gating mechanism of the large mechanosensitive channel MscL; Sukharev S et al.; The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria . The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating . Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure . When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker . We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation . Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening . S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing . Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.

Chem Pharm Bull (Tokyo), 2001 Feb, 49(2), 197 - 202
Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus . Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis; Sitthithaworn W et al.; cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method . Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence . When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP) . The structural factors determining the product length in plant GGPPSs were investigated by constructing S . dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase . The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length . Further, when a chimeric gene comprised of the putative transit peptide of the S . dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S . dulcis GGPPS is a chloroplast protein.

J Vet Med Sci, 2001 Jan, 63(1), 61 - 6
Analysis by enzyme-linked immunosorbent assay and 2-dimensional electrophoresis of haptoglobin in the high-density lipoprotein fraction in cows; Kanno H et al.; Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein . Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism . In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions . The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis . When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml . The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884 . The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions . Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 microg of Hp/ml (n = 4), and concentration ratios were 0.2 to 0.3% of whole serum Hp . Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction . By 2-dimensional electrophoresis, alpha- and beta-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the alpha-chain and from 5.92 to 6.95 in the beta-chain . The pI values of HDL Hp were indistinguishable from those of serum Hp . These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction.

J Environ Qual, 2001 Jan-Feb, 30(1), 171 - 9
Using zebra mussels to monitor Escherichia coli in environmental waters; Selegean JP et al.; Use of the zebra mussel (Dreissena polymorpha) as an indicator of previously elevated bacteria concentrations in a watershed was examined . The ability of the zebra mussel to accumulate and purge Escherichia coli over several days was investigated in both laboratory and field experiments . In laboratory experiments, periodic enumeration of E . coli in mussels that had been exposed to a dilute solution of raw sewage demonstrated that (i) maximum concentrations of E . coli are reached within a few hours of exposure to sewage, (ii) the tissue concentration attained is higher than the concentration in the ambient water, and (iii) the E . coli concentrations take several days to return to preexposure concentrations when mussels are subsequently placed in sterile water . In field experiments conducted in southeast Michigan in the Clinton River watershed, brief increases in E . coli concentrations in the water were accompanied by increases in mussel concentrations of E . coli that lasted 2 or 3 d . The ability of mussels to retain and to concentrate E . coli made it possible to detect E . coli in the environment under conditions that conventional monitoring may often miss . Sampling caged mussels in a river and its tributaries may enable watershed managers to reduce the sampling frequency normally required to identify critical E . coli sources, thereby providing a more cost-effective river monitoring strategy for bacterial contamination.

Hum Genet, 2001 Jan, 108(1), 37 - 42
Human type I hair keratin pseudogene phihHaA has functional orthologs in the chimpanzee and gorilla: evidence for recent inactivation of the human gene after the Pan-Homo divergence; Winter H et al.; In addition to nine functional genes, the human type I hair keratin gene cluster contains a pseudogene, phihHaA (KRTHAP1), which is thought to have been inactivated by a single base-pair substitution that introduced a premature TGA termination codon into exon 4 . Large-scale genotyping of human, chimpanzee, and gorilla DNAs revealed the homozygous presence of the phihHaA nonsense mutation in humans of different ethnic backgrounds, but its absence in the functional orthologous chimpanzee (cHaA) and gorilla (gHaA) genes . Expression analyses of the encoded cHaA and gHaA hair keratins served to highlight dramatic differences between the hair keratin phenotypes of contemporary humans and the great apes . The relative numbers of synonymous and non-synonymous substitutions in the phihHaA and cHaA genes, as inferred by using the gHaA gene as an outgroup, suggest that the human hHaA gene was inactivated only recently, viz., less than 240,000 years ago . This implies that the hair keratin phenotype of hominids prior to this date, and after the Pan-Homo divergence some 5.5 million years ago, could have been identical to that of the great apes . In addition, the homozygous presence of the phihHaA exon 4 nonsense mutation in some of the earliest branching lineages among extant human populations lends strong support to the "single African origin" hypothesis of modern humans.

Environ Microbiol, 2000 Dec, 2(6), 594 - 610
Population genetics of Escherichia coli in a natural population of native Australian rats; Pupo GM et al.; Escherichia coli, a normal inhabitant of the intestinal tract of mammals and birds, is a diverse species . Most studies on E . coli populations involve organisms from humans or human-associated animals . In this study, we undertook a survey of E . coli from native Australian mammals, predominantly Rattus tunneyi, living in a relatively pristine environment in the Bundjalung National Park . The genetic diversity was assessed and compared by multilocus enzyme electrophoresis (MLEE), sequence analysis of the mdh (malate dehydrogenase) gene and biotyping using seven sugars . Ninety-nine electrophoretic types were identified from the 242 isolates analysed by MLEE and 15 sequences from the mdh genes sequenced from 21 representative strains . The Bundjalung isolates extend the diversity represented by the E . coli reference (ECOR) set, with new MLEE alleles found in six out of 10 loci . Many of the Bundjalung isolates fell into a discrete group in MLEE . Other Bundjalung strains fell into the recognized E . coli ECOR set groups, but tended to be at the base of both the MLEE and mdh gene trees, implying that these strains are derived independently from ancestral forms of the ECOR groups and that ECOR strains represent only a subset of E . coli adapted to humans and human-associated animals . Linkage disequilibrium analysis showed that the Bundjalung population has an 'epidemic' population structure . The Bundjalung isolates were able to utilize more sugars than the ECOR strains, suggesting that diet plays a prominent role in adaptation of E . coli.

Poult Sci, 2001 Jan, 80(1), 22 - 8
DNA microsatellites linked to quantitative trait loci affecting antibody response and survival rate in meat-type chickens; Yonash N et al.; Selection for immune response parameters may lead to improved general disease resistance . Because disease resistance and immune response are hard-to-measure quantitative traits with low to moderate heritability, they may respond more efficiently to marker-assisted selection (MAS) than to phenotypic selection . To detect DNA markers linked to quantitative trait loci (QTL) associated with immune response, a resource half-sib family of 160 backcross (BC1) and intercross (F2) birds was derived from a cross between two meat-type lines divergently selected for high or low antibody (Ab) response to Escherichia coli . By using 25 microsatellite DNA markers covering approximately 25% of the chicken genome, initial genotyping of 40% of the resource family was followed by complete genotyping of the entire family with four suggestive markers . Three of these markers exhibited significant association with immune response: (1) ADL0146 on Chromosome 2 associated with Ab to SRBC and Newcastle disease virus (NDV), (2) ADL0290 on linkage group 31 affecting Ab to NDV, and (3) ADL0298 on linkage group 34 associated with Ab to E . coli and survival . The family was also genotyped with five linked markers from two of the suggested regions, and interval mapping was applied . The results confirmed the significant effects, suggested the location of the QTL, and confirmed the genetic association between immune responses and disease resistance . These findings support the idea of improving poultry immunocompetence by MAS.

Dev Biol (Basel), 2000, 103, 163 - 73
Characterization of a recombinant outer surface protein A (OspA) vaccine against Lyme disease; Huebner RC et al.; Lyme disease, the most prevalent tick-borne disease in the United States, results from infection with Borrelia burgdorferi sensu stricto . Early studies of Borrelia burgdorferi sensu stricto identified outer surface protein A (OspA), a lipoprotein on the surface of spirochetes that could be the target of protective antibodies to this agent . Pasteur Merieux Connaught has developed a Lyme vaccine, ImuLyme, using recombinant OspA protein (rOspA) . Methods were developed to routinely assess the identity, quantity, structure, purity, biological activity, heterogeneity, stability, and potency of rOspA . In addition, several methods were performed on a series of lots to support the routine testing methods and further our understanding of the physicochemical characteristics of rOspA . These tests were electrospray mass spectrometry, circular dichroism, two-dimensional gel electrophoresis, amino acid analysis, peptide mapping with peptide sequencing, and the application of proteomic methodology to identify trace contaminant host cell proteins . The results of these methods indicate that the rOspA lots are composed of highly purified and properly processed and folded rOspA with trace amounts of E . coli host cell proteins.

Clin Lab, 2001, 47(1-2), 19 - 27
Recombinant single chain cardiac troponin I-C polypeptides: superior calibration and control materials for cardiac troponin I immunoassays; Liu S et al.; There has been a need to create stable and reproducible calibration and control materials for cardiac troponin I assays . Free troponin I, native or recombinant, has been known to be unstable, while troponin CI complex can be easily dissociated in low concentrations or in the presence of chelating agents . In order to overcome these difficulties, two single chain troponin I-C polypeptides have been engineered and expressed separately in Escherichia coli . One consists of a full-length of human cardiac troponin I and C, termed as ScTnI-C and the other consists of a stable fragment (aa28-110) of human cardiac troponin I and a full-length troponin C, termed as ScTnI-C-2 . Both ScTnI-C and ScTnI-C-2 were purified to homogeneity by affinity chromatography using anti-cTnI monoclonal antibodies . ScTnI-C and ScTnI-C-2 have apparent molecular weights of 45 kD and 30 kD by SDS-PAGE, respectively . Stability studies by Stratus showed that ScTn I-C and ScTnI-C-2 were stable for 4 months at 2-8 degrees C and at least one year at -20 degrees C . When incubated in human serum at 37 degrees C, ScTnI-C-2 was more resistant to proteolysis than ScTnI-C . ScTnI-C can be recognized by all commercial TnI immunoassays with excellent activity . ScTnI-C-2 can be recognized by all immunoassays that target the stable region of cardiac troponin I . Judging by their performances, ScTnI-C and ScTnI-C-2 are both superior materials to be used as calibrators and controls in clinical laboratories.

RNA, 2001 Jan, 7(1), 71 - 84
Arrangement of the central pseudoknot region of 16S rRNA in the 30S ribosomal subunit determined by site-directed 4-thiouridine crosslinking; Juzumiene DI et al.; The 16S rRNA central pseudoknot region in the 30S ribosomal subunit has been investigated by photocrosslinking from 4-thiouridine (s4U) located in the first 20 nt of the 16S rRNA . RNA fragments (nt 1-20) were made by in vitro transcription to incorporate s4U at every uridine position or were made by chemical synthesis to incorporate s4U into one of the uridine positions at +5, +14, +17, or +20 . These were ligated to RNA containing nt 21-1542 of the 16S rRNA sequence and, after gel purification, the ligated RNA was reconstituted into 30S subunits . Long-range intramolecular crosslinks were produced by near-UV irradiation; these were separated by gel electrophoresis and analyzed by reverse transcription reactions . A number of crosslinks are made in each of the constructs, which must reflect the structural flexibility or conformational heterogeneity in this part of the 30S subunit . All of the constructs show crosslinking to the 559-562, 570-571, and 1080-1082 regions; however, other sites are crosslinked specifically from each s4U position . The most distinctive crosslinking sites are: 341-343 and 911-917 for s4U(+5); 903-904 (very strong), 1390-1397, and 1492 for s4U(+14); and 903-904 (moderate) for s4U(+17); in the 1070-1170 region in which there are different patterns for each s4U position . These results indicate that part of the central pseudoknot is in close contact with the decoding region, with helix 27 in the 885-912 interval and with part of domain III RNA . Crosslinking between s4U(+14) and 1395-1397 is consistent with base pairing at U14-A1398.

RNA, 2001 Jan, 7(1), 64 - 70
Functional mapping of ribosome-contact sites in the ribosome recycling factor: a structural view from a tRNA mimic; Fujiwara T et al.; Ribosome recycling factor (RRF) is required for disassembly of the posttermination complex of the ribosome after release of polypeptides . The crystal structure of RRF resembles a tRNA shape, with an architecturally different flexibility compared with tRNA, but its structure-and-function relationships are unknown . We here found that an RRF variant defective in ribosome binding regains the binding capacity through 20 independent secondary changes occurring in three topologically distinct regions of RRF . Because two of these regions are equivalent to the tip of the anticodon stem and the upper surface of the acceptor stem of tRNA, RRF may interact with the ribosome in a way similar to tRNA, spanning 30S and 50S subunits, to exert its action for splitting the ribosome.

Antioxid Redox Signal, 2000 Winter, 2(4), 811 - 20
Antioxidant function of thioredoxin and glutaredoxin systems; Holmgren A; Selenium is an essential trace element with known antioxidant properties . Cytosolic thioredoxin reductase from mammalian cells is a dimeric flavin enzyme comprising a glutathione reductase-like equivalent elongated with 16 residues including the conserved carboxy-terminal sequence, Gly-Cys-SeCys-Gly, where SeCys is selenocysteine . Replacement of the SeCys residue by Cys in rat cytosolic thioredoxin reductase using site-directed mutagenesis and expression in Escherichia coli resulted in a functional mutant enzyme having about one percent activity with thioredoxin as a substrate through a major loss of Kcat and a shift in the pH optimum from 7 to 9 . The truncated enzyme expected in selenium deficiency by the UGA mRNA codon for SeCys acting as a stop codon was also expressed . This enzyme lacking the carboxy-terminal SeCys-Gly dipeptide contained FAD but was inactive because the SeCys selenol is in the active site . These results show that selenium is essential for the activity of thioredoxin reductase, explaining why this trace element is required for cell proliferation by effects on thioredoxin-dependent control of the intracellular redox state, ribonucleotide reductase production of deoxyribonucleotides, or activation of transcription factors . The selenazol drug ebselen (2-phenyl-1,2 benzisoselenazol-3 (2H)-one) is a known glutathione (GSH) peroxidase mimic with antioxidant properties . The hydrogen peroxide reductase activity of human thioredoxin reductase was stimulated 15-fold by 2 microM ebselen . Glutaredoxins protect against oxidative stress by catalyzing reduction of protein mixed disulfides with GSH . The mechanism of glutaredoxins as efficient general GSH-mixed disulfide oxidoreductases may protect proteins from inactivation as well as play a major role in general redox signaling.

Int J Radiat Biol, 2001 Jan, 77(1), 53 - 8
Mutagenic effects of 5-formyluracil on a plasmid vector during replication in Escherichia coli; Miyabe I et al.; PURPOSE: 5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants . It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis . The present experiments were designed to understand the biological effects of5-foU in vivo . MATERIALS AND METHODS: The modified base was incorporated site-specifically into the recognition site of restriction endonuclease SalI (5'-GTCGAC) or AflII (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli . RESULTS: When the plasmids were replicated in E . coli, 5-foU caused mutations at the target sites . The induced mutation frequencies were 0.038-0.049% . Sequence analysis revealed that 5-foU preferentially caused T:A-->C:G and T:A-->A:T base substitutions and -1 deletions at the 5-foU site . 5-FoU also caused mutations at sites near the 5-foU . The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids . CONCLUSIONS: The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E . coli.

Mol Gen Genet, 2001 Jan, 264(5), 709 - 15
An Aspergillus nidulans uvsC null mutant is deficient in homologous DNA integration; Ichioka D et al.; The Aspergillus nidulans uvsC gene was identified as a homolog of RAD51 and recA of Saccharomyces cerevisiae and Escherichia coli, respectively, whose role in genetic recombination and recombinational repair has been extensively studied . Like many other filamentous fungi, A . nidulans shows no bias towards either homologous or ectopic integration of exogenous DNA . Therefore it is a unique and useful organism for the study of the mechanisms of DNA integration . Homologous integration of a 1.7-kb argB gene was not detected in 50 transformants obtained from a uvsC null mutant . In contrast, the frequency of homologous integration in uvsC+ control strains varied from 41 to 86% . Another feature observed with the uvsC null mutant was that an increased number of transformants had undergone ectopic integrations at multiple sites in the genome . These results are consistent with the established function of Rad51/RecA, and further indicate the involvement of redundant pathways in integration of exogenous DNA . This study provides direct evidence for the involvement of uvsC in exogenous DNA integration and should contribute to the improvement of genetic manipulations in general, but particularly in fungi.

Mol Gen Genet, 2001 Jan, 264(5), 674 - 81
Two novel genes encoding SNF-1 related protein kinases from Arabidopsis thaliana: differential accumulation of AtSR1 and AtSR2 transcripts in response to cytokinins and sugars, and phosphorylation of sucrose synthase by AtSR2; Chikano H et al.; We searched for genes encoding members of the group-3 SNF1-related protein kinase (SnRK3) family in the Arabidopsis thaliana database, and seven independent sequences were identified . Transcripts of two of them were found to accumulate differentially upon treatment with light, cytokinins and sugars . Full-length cDNAs were isolated and designated as AtSR1 and AtSR2; they encode polypeptides of 442 and 429 amino acids with relative molecular masses of 50.3 kDa and 48.2 kDa, respectively . In etiolated seedlings, no transcripts of either gene were observed . However, upon exposure to light or cytokinins, transcripts of AtSR1 but not AtSR2 began to accumulate . The induction with light was greatly reduced in the presence of a cytokinin antagonist, suggesting that cytokinins are involved in light-signaling pathways . In contrast, transcription of AtSR2, but not of AtSR1, was greatly increased by sucrose, as well as glucose and fructose . AtSR2 expressed in E . coli efficiently phosphorylated sucrose synthase in the presence of manganese ions . These results suggest that, although SnRK3 proteins may generally be involved in sugar metabolism, expression of AtSR1 and AtSR2 is differentially and distinctly regulated by various external signals, and AtSR2 may function in the regulation of sucrose synthase by specific phosphorylation.

Mol Gen Genet, 2001 Jan, 264(5), 578 - 87
A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP; Wang Y et al.; We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine . In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC) . The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase . The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry . The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry . Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Mol Gen Genet, 2001 Jan, 264(5), 531 - 8
Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression; Sujatha S et al.; Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase . The mutant alpha-subunit C131A is unable to complement the temperature-sensitive alpha-R45C mutant strain, which is defective for binding of the beta-subunit . In vitro reconstitution experiments, however, indicate that the alpha-C131A variant is able to form the intermediate alpha2beta, but is defective in contacting the beta'-subunit . We used this alpha-C131A mutant to isolate a suppressor mutation in the beta'-subunit . Genetic and biochemical characterization of the beta' suppressor indicates the allele-specific nature of its effect . Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the beta'-subunit, by Asp . However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (alpha-C131A) and its suppressor lie far apart . Thus, we propose that long-range interactions, as in this case, may play an important role in the functional assembly of E . coli RNA polymerase.

Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 817 - 24
The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore; Meisinger C et al.; Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex) . This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane . This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.

Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 735 - 41
Evolution of virulence factors in Shiga-toxin-producing Escherichia coli; Boerlin P; The major demonstrated or putative virulence factors of Shiga-toxin-producing Escherichia coli (STEC) are the Shiga toxins, products of the locus of enterocyte effacement, and products encoded by the EHEC-hemolysin plasmid . Molecular analysis shows that STEC acquired the majority of these virulence factors by horizontal transfer of genetic material . In the case of Shiga toxins, the phages encoding them are probably responsible for this transfer . For the locus of enterocyte effacement, however, it is not clear how often this transfer took place and which parts of the locus were involved in this transfer . The large EHEC-hemolysin plasmid is clearly a mosaic structure, which arose from multiple recombination events with foreign DNA . Two lineages of this plasmid can be distinguished, one of which is associated with chromosomally encoded virulence factors . Despite the wealth of information available, further comparative studies are needed to decipher definitively the evolution of virulence in STEC.

Cell Mol Life Sci, 1999 Oct 30, 56(5-6), 507 - 22
Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product; Holden HM et al.; Carbamoyl phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry, in which carbamoyl phosphate is produced from one molecule of bicarbonate, two molecules of Mg2+ ATP, and one molecule of either glutamine or ammonia . The carbamoyl phosphate so produced is utilized in the synthesis of arginine and pyrimidine nucleotides . It is also employed in the urea cycle in most terrestrial vertebrates . Due to its large size, its important metabolic role, and the fact that it is highly regulated, CPS has been the focus of intensive investigation for nearly 40 years . Numerous enzymological, biochemical, and biophysical studies by a variety of investigators have led to a quite detailed understanding of CPS . Perhaps one of the most significant advances on this topic within the last 2 years has been the successful X-ray crystallographic analysis of CPS from Escherichia coli . Quite unexpectedly, this structural investigation revealed that the three active sites on the protein are widely separated from one another . Furthermore, these active sites are connected by a molecular tunnel with a total length of approximately 100 A, suggesting that CPS utilizes this channel to facilitate the translocation of reaction intermediates from one site to another . In this review, we highlight the recent biochemical and X-ray crystallographic results that have led to a more complete understanding of this finely tuned instrument of catalysis.

Cancer Res, 2001 Jan 15, 61(2), 666 - 72
Creation and characterization of 5-fluorodeoxyuridine-resistant Arg50 loop mutants of human thymidylate synthase; Landis DM et al.; Thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA . Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy . In the cell, 5-FU is metabolized to 5-fluoro-2'-deoxyuridine 5'-monophosphate, a tight binding covalent inhibitor of thymidylate synthase . Recent studies have identified 5-fluoro-2'-deoxyuridine (5-FdUR) and antifolate-resistant mutants of human thymidylate synthase (TS) that contain single residue substitutions within the highly conserved Arg50-loop, which binds the pyrimidine substrate (Y . Tong et al., J . Biol . Chem . 273: 11611-11618, 1998) . We have used random sequence mutagenesis to gain structure-function information about the TS and to create novel drug-resistant mutants for gene therapy . A library of 1.5 million mutants of the Arg50-loop and the nearby residue Tyr 33 was selected to identify mutants of the human enzyme with the ability to complement a thymidylate synthase-deficient Escherichia coli strain and form colonies in the presence of 5-FdUR . E . coli-harboring plasmids that were encoding TS with single, double, and triple amino acid substitutions were identified that survive at dosages of 5-FdUR clearly lethal to E . coli harboring either wild-type thymidylate synthase or constructs encoding previously characterized drug resistant mutants . Four 5-FdUR-resistant mutants were purified to apparent homogeneity . Kinetic studies indicate that these enzymes are highly efficient . Inhibition constants (Ki) for the double mutant K47Q;D48E and the triple mutant D48E;T51S;G52C in the presence of 5-fluoro-2'-deoxyuridine 5'-monophosphate were determined to be 75 to 100 times higher, respectively, than that of the wild-type enzyme . These mutant TSs, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of 5-FU chemotherapy.

Cancer Res, 2001 Jan 15, 61(2), 478 - 81
Soluble recombinant endostatin purified from Escherichia coli: antiangiogenic activity and antitumor effect; Huang X et al.; Endostatin is a potent and specific antiangiogenic protein capable of inhibiting the growth of murine and xenotransplanted human tumors . Thus far, however, recombinant endostatin prepared from Escherichia coli is insoluble after purification and therefore inappropriate for clinical settings . A soluble form of endostatin is available from a yeast system with relatively low yield and high cost, which has made it difficult to produce endostatin in quantities sufficient for extensive clinical evaluation . In this study, we developed a protocol to generate soluble recombinant murine endostatin from E . coli at a yield of 150 mg/liter-culture and 99% purity . The in vivo antiangiogenic and antitumor activities of the soluble recombinant endostatin are equally as potent as those of the previously published insoluble form . A similar protocol may be used to produce soluble human endostatin.

Mol Cells, 2000 Dec 31, 10(6), 684 - 91
Expression and characterization of fibroblast growth factor 8 from Mexican axolotl, Ambystoma mexicanum; Lee SY et al.; Fibroblast growth factor (FGF) has been known to regulate the proliferation and differentiation of a variety of cell types via interaction with a specific FGF receptor on the cell surface . In the present study, Fgf8 cDNA of Mexican axolotl, Ambystoma mexicanum, was expressed in Escherichia coli as an MBP-FGF8 fusion protein . The cell proliferation activity of the recombinant FGF8 (rFGF8) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay . The addition of rFGF8 to the culture medium enhanced proliferation of BALB/c 3T3 and BHK21 cells about 1.4-1.5 fold . To analyze the binding activity of rFGF8 to the cell surface, cell surface enzyme linked immunosorbent assay was developed . Comparison of the structure of basic FGF with the computer-simulated structure of FGF8 suggested that Tyr-58, Glu-132, Tyr-139, and Leu-179 might be the potential receptor binding sites . Amino acid substitution muteins of FGF8 were constructed by PCR-derived directed mutagenesis and the muteins were overexpressed in E . coli . The rFGF8 muteins were purified and their binding activities were analyzed . Substitution of Tyr-58 or Glu-132 or Leu-179 of the FGF8 with alanine reduced the binding affinity, while substitution of Tyr-139 with alanine did not alter the binding affinity . These results imply that Tyr-58, Glu-132, and Leu-179 of FGF8 might be involved in its binding to the cell surface.

Mol Cells, 2000 Dec 31, 10(6), 662 - 8
Gene delivery into rat glomerulus using a mesangial cell vector; Kim HJ et al.; To develop an effective protocol of gene transfer into glomeruli, an ex vivo gene delivery system using rat mesangial cells (RMC) as a vector was examined . RMC genetically engineered with a retrovirus harboring the Escherichia coli beta-galactosidase gene was used to estimate the efficacy of gene delivery and the location of the cells within the kidney . The RMC expressing beta-galactosidase, RMCLZ1, was cultured in vitro and the cells were injected into the left kidney through the renal artery of a normal Sprague Dawley rat . At least 1 x 10(6) RMCLZ1 was required for effective gene delivery into glomeruli . One hour and 1, 4, and 14 d after injection, glomeruli were isolated from the left kidneys injected with the cells and the expression of beta-galactosidase in each glomeruli was evaluated . One hour and 1 d after injection, more than 90 and 80%, respectively, of glomeruli from the left kidney showed strong beta-galactosidase activity, while no activity of beta-galactosidase was found in the glomeruli from the right kidneys . The number of glomeruli stained by X-gal and the intensity decreased with time . Fourteen days after injection, about 35% of the glomeruli retained the RMCLZ1 . X-gal and periodic acid-Schiff staining of frozen sections obtained 14 d after injection allowed the estimation of the site where the mesangial cells injected were located . The mesangial cells were found mainly in two different locations, the glomerular capillary and the mesangium . The majority (about 90%) of the mesangial cells were located in the glomerular capillary and about 9% of the cells were in the mesangial area . Occasionally, the positive staining was found in proximal tubules and the interlobular artery . Although additional methods are required for the site-specific targeting of the mesangial area, the ex vivo gene transfer to glomeruli is feasible and may be a useful tool for future investigations in the pathological mechanisms of glomerular injury.

Mol Cells, 2000 Dec 31, 10(6), 633 - 41
Cloning and characterization of a 22 kDa outer-membrane protein (Omp22) from Helicobacter pylori; Kim JS et al.; Helicobacter pylori is a causative agent of gastritis and peptic ulceration in humans . As the first step towards development of a vaccine against H . pylori infection, we have attempted to identify protective antigens . A potential target of vaccine development would be a H . pylori specific protein, which is surface-exposed and highly antigenic . We identified a 22 kDa outer-membrane protein (Omp22) from H . pylori, which was highly immunoreactive . By screening a H . pylori genomic DNA library with rabbit anti-H . pylori outer-membrane protein antibodies, the omp22 gene was cloned and 1.4 kb of the nucleotide sequence was determined . One open reading frame, encoding a 179-residue polypeptide, was identified and the amino acid sequence deduced showed homology with peptidoglycan-associated lipoproteins . The sequence was conserved among other H . pylori strains . Omp22 protein is expressed as a precursor polypeptide of 179 residues and undergoes lipid modification and cleavage of an 18 amino acid signal peptide to yield a mature protein . Omp22 protein in H . pylori as well as recombinant Omp22 protein expressed in E . coli was localized into the outer membrane and exposed on the cell surface . Omp22 may have the potential as a target antigen for the development of a H . pylori vaccine.

Hum Exp Toxicol, 2000 Nov, 19(11), 604 - 14
Effect of natural antioxidants and apocynin on LPS-induced endotoxemia in rabbit; Ben-Shaul V et al.; The objective of this study was to compare the prophylactic effects of the natural antioxidant from spinach (NAO) and apocynin, on the hepatic oxidative stress and liver damage induced by lipopolysaccharide (LPS) . Male New Zealand rabbits were challenged with LPS with or without 8 days of antioxidant pretreatment . Pretreatment with NAO, but not apocynin, significantly (p < 0.05) decreased the levels of hydroperoxides and malondialdehyde (MDA) in the liver cytosolic fraction and the activity of NADPH oxidase-generated superoxide in the microsomal fraction, compared to LPS alone . The activity of glutathione peroxidase (G-POX) was significantly (p < 0.05) increased in the LPS-treated group, whereas treatment with NAO, but not apocynin, significantly (p < 0.05) decreased G-POX activity . Pretreatment with the same antioxidants had no significant effects on superoxide dismutase (SOD) activity, whereas an increased level of catalase (CAT) was obtained in all LPS-treated groups . TUNEL immunohistochemical staining in the LPS-treated animals indicated that there was no increase in apoptosis outside of necrotic foci . However, apoptotic hepatocytes were observed within areas of focal necrosis in animals exposed to LPS alone or LPS plus apocynin . Hepatocyte cell proliferation was tested by the proliferating-cell nuclear antigen (PCNA) tool, which indicated a proliferative effect in the LPS group, whereas the effect disappeared in the antioxidant-treated groups . The prophylactic effect of NAO on liver pathology and the significant decreases in lipid peroxidation products and NADPH oxidase activity suggest the use of NAO as an efficient strategy for treatment of endotoxemia.

Mamm Genome, 2001 Feb, 12(2), 133 - 40
The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1; Fletcher BH et al.; Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties . Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone . The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes . In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence . To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF . Eight distinct genes were identified . Cpn10 contains introns, while other members are intronless . Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein . The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes . Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR . Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression . Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively . The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF . The complete ORF of Cpn10-rs1 was expressed in E . coli . The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

Biosci Biotechnol Biochem, 2000 Dec, 64(12), 2731 - 3
Introduction of enterostatin (VPDPR) and a related sequence into soybean proglycinin A1aB1b subunit by site-directed mutagenesis; Takenaka Y et al.; Enterostatin (VPDPR), having anoretic and hypocholesterolemic activities, and its homologue LPYPR, a hypocholesterolemic peptide found in the glycinin A5A4B3 subunit, were introduced into the corresponding site (TNGPQ) of the proglycinin A1aB1b subunit by site-directed mutagenesis . Modified proglycinins were expressed in E . coli and recovered from the insoluble fraction . VPDPR and LPYPR were released by the action of chymotrypsin and trypsin as expected . The overall yields of purified VPDPR and LPYPR were 40% and 62%, respectively.

Biol Chem, 2000 Dec, 381(12), 1251 - 8
Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen; Greenbaum L et al.; Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP) . The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E . coli bacteria following light irradiation . Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6 . A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6 . However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching . Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy . Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles . Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles . We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.

Biol Chem, 2000 Dec, 381(12), 1245 - 9
Recombinant anti-stefin A Fab fragment: sequence analysis of the variable region and expression in Escherichia coli; Kopitar-Jerala N et al.; Human stefin A is an inhibitor of lysosomal cysteine proteinases cathepsin B, H, L and S . In the present report we describe the cloning and expression of anti-stefin A Fab fragment A22 in E . coli . We have determined the nucleotide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences . Expression of the two antibody chains was achieved using a single vector with a PhoA promoter and coding regions placed after the signal sequences, directing them to the periplasmic space . The A22 Fab fragment was extracted from the periplasmic space and expression was confirmed by Western blot analysis . The recombinant A22 Fab fragment had an affinity for stefin A comparable to the original monoclonal antibody, as determined by ELISA.

Yi Chuan Xue Bao, 2000, 27(11), 1006 - 11
{Expression of single-chain Fv antibody for anti-beet necrotic yellow vein virus in Escherichia coli}; Zeng JZ et al.; The heavy chains variable region gene (VH) of monoclonal antibody against beet necrotic yellow vein virus (BNYVV) was amplified from total DNA extracted from anti-BNYVV hybridoma cells by PCR . Sequencing showed that the VH belongs to mouse subgroup II(A) and contains 360 bp, which code one hundred and twenty amino acids . The VH and VL genes were inserted into a plasmid which contains a linker sequence for constructing scFv gene . The new vector named pTC scFv . The scFv was produced in Escherichia coli and appeared binding activity with BNYVV antigen by ELISA method.

Fundam Clin Pharmacol, 2000 Nov-Dec, 14(6), 593 - 600
Effect of lipopolysaccharide treatment on neurogenic contraction and noradrenaline release in rat arteries; Ohlmann P et al.; In the present study, contractile responses and {3H}-noradrenaline overflow evoked by electrical field stimulation were assessed, respectively, in the small mesenteric artery and in tail artery removed from rats pre-treated with either saline or lipopolysaccharide (LPS) . In small mesenteric arteries, LPS treatment did not significantly modify the contractile responses elicited by electrical stimulation, in the absence or in the presence of L-arginine . However, in arteries removed from rats treated with LPS, L-arginine addition produced relaxation of vessels pre-contracted with noradrenaline . The amplification of neurogenic contraction by the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) was similar in arteries removed from saline and LPS-infused rats . In mesenteric arteries, LPS treatment suppressed the potentiation of the neurogenic responses by the alpha2-adrenoceptor antagonist, yohimbine and by the inhibitor of neuronal uptake of noradrenaline, cocaine . In rat tail artery exposed to L-arginine, LPS treatment produced an increase in {3H}-noradrenaline overflow evoked by electrical stimulation . Altogether, these data suggest that an enhanced noradrenaline release from sympathetic nerves, probably resulting from inhibition of the modulatory effect of both prejunctional alpha2-adrenoceptors and neuronal uptake mechanism, may play a role in the preservation of neurogenic response after LPS treatment despite evidence of the induction of NO synthase.

Bioorg Med Chem Lett, 2001 Jan 22, 11(2), 119 - 22
Which aminoglycoside ring is most important for binding? A hydropathic analysis of gentamicin, paromomycin, and analogues; Cashman DJ et al.; The NMR structures of gentamicin and paromomycin in complex with the A-site of Escherichia coli 16S ribosomal RNA were modified with molecular modeling to 12 analogues . The intermolecular interactions between these molecules and RNA were examined using the HINT (Hydropathic INTeractions) computational model to obtain interaction scores that have been shown previously to be related to free energy . The calculations correlated well with experimental binding data, and the interaction scores were used to analyze the specific structural features of each aminoglycoside that contribute to the overall binding with the 16S rRNA . Our calculations indicate that, while ring I binds to the main binding pocket of the rRNA A-site, ring IV of paromomycin-based aminoglycosides contributes significantly to the overall binding.

J Gastroenterol Hepatol, 2001 Jan, 16(1), 61 - 9
Endotoxin treatment causes an upregulation of the endothelin system in the liver: amelioration of increased portal resistance by endothelin receptor antagonism; Gandhi CR et al.; BACKGROUND: Mechanisms underlying hepatic microcirculatory failure during endotoxemia are incompletely understood . Because endothelin-1 (ET-1) has been implicated in endotoxin-induced liver injury, we investigated the hepatic ET-1 system in endotoxin-treated rats . METHODS: Rats were treated with endotoxin (Escherichia coli lipopolysaccharide; 3 mg/kg, i.p.), and various determinations were made 24 h later . RESULTS: Endotoxin treatment caused 11.2 +/- 1.6% weight loss, a decrease in mean arterial pressure (MAP; 96 +/- 5 mmHg vs 108 +/- 3 mmHg; P < 0.05) and an increase in portal pressure (11.6 +/- 1.3 mmHg vs 7.4 +/- 1 mmHg; P < 0.02) . No significant changes in the serum levels of liver enzymes or hepatocellular necrosis were observed . Endotoxin caused increases in hepatic ET-1 (from 345 +/- 31 to 565 +/- 38 pg/g; P < 0.01), ET-1 receptor density (from 179 +/- 16 to 340 +/- 26 fmol/mg; P < 0.02), and mRNA expression of preproendothelin-1, and ET(A) and ET(B) receptors . While the serum nitric oxide (nitrite +/- nitrate) concentration was increased in endotoxin-treated rats, that of ET-1 remained unchanged . A mixed ET(A)/ET(B) receptor antagonist, TAK-044 (10 mg/kg, i.v.), reduced the weight loss from 11.2 +/- 1.6% to 5.9 +/- 2.9% (P < 0.05) and the portal pressure from 11.6 +/- 1.3 mmHg to 8.6 +/- 0.7 mmHg (P < 0.05) in endotoxin-treated rats . The mixed ET(A)/ET(B) receptor antagonist also caused an increase in serum ET-1 concentration, but did not affect serum nitric oxide and MAP in endotoxin-treated rats . CONCLUSIONS: These results suggest that the upregulated hepatic ET-1 system is an important mechanism of increased portal resistance and related complications of endotoxemia.

Parasitol Res, 2001 Feb, 87(2), 112 - 5
Characterization of a proteasome alpha-chain from Giardia lamblia; Emmerlich V et al.; To begin to characterize the components of the 20S proteasome of Giardia lamblia, we have cloned a genomic sequence encoding an alpha-chain (type alpha3/C9, predicted size 244 amino acid residues) . Southern analysis indicated that a single gene codes for this protein, and a Northern blot exhibited a single signal at 850 nt . An antiserum against a C-terminal fragment of the alpha-chain expressed in Escherichia coli reacted with a single protein band of Mr 27,000 that was present at constant levels in trophozoites and encysting cells . On a 2D blot of the purified 20S proteasome, we identified the cross-reacting component as a single protein of IEP 6.0, in agreement with the IEP predicted by the coding sequence . Our data confirm that the G . lamblia 20S proteasome is typically eukaryotic in containing a set of diverged alpha-subunits.

Protein Sci, 2000 Dec, 9(12), 2573 - 6
Fluorescence and 19F NMR evidence that phenylalanine, 3-L-fluorophenylalanine and 4-L-fluorophenylalanine bind to the L-leucine specific receptor of Escherichia coli; Luck LA et al.; The binding capacity of the L-leucine receptor from Escherichia coli was measured with L-phenylalanine and 4-fluoro-L-phenylalanine as substrates by fluorescence . The apparent dissociation constants (KD) for L-leucine, L-phenylalanine, and 4-fluoro-L-phenylalanine are 0.40, 0.18, and 0.26 respectively . 19F NMR data show protein-induced shifts for the 4-fluoro-L-phenylalanine peak and 3-fluoro-L-phenylalanine when receptor is present . Evidence points to the binding of only the L-isomers of these fluorine analogs.

Protein Sci, 2000 Dec, 9(12), 2557 - 66
The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding; Teplova M et al.; The yrdC family of genes codes for proteins that occur both independently and as a domain in proteins that have been implicated in regulation . An example for the latter case is the sua5 gene from yeast . SuaS was identified as a suppressor of a translation initiation defect in cytochrome c and is required for normal growth in yeast (Na JG, Pinto I, Hampsey M, 1992, Genetics 11:791-801) . However, the function of the Sua5 protein remains unknown; Sua5 could act either at the transcriptional or the posttranscriptional levels to compensate for an aberrant translation start codon in the cyc gene . To potentially learn more about the function of YrdC and proteins featuring this domain, the crystal structure of the YrdC protein from Escherichia coli was determined at a resolution of 2.0 A . YrdC adopts a new fold with no obvious similarity to those of other proteins with known three-dimensional (3D) structure . The protein features a large concave surface on one side that exhibits a positive electrostatic potential . The dimensions of this depression, its curvature, and the fact that conserved basic amino acids are located at its floor suggest that YrdC may be a nucleic acid binding protein . An investigation of YrdC's binding affinities for single- and double-stranded RNA and DNA fragments as well as tRNAs demonstrates that YrdC binds preferentially to double-stranded RNA . Our work provides evidence that 3D structures of functionally uncharacterized gene products with unique sequences can yield novel folds and functional insights.

Protein Sci, 2000 Dec, 9(12), 2405 - 12
Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement; Voziyan PA et al.; One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation . Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold . We have found that the refolding of glutamine synthetase (GS) does not follow this pattern . In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration . Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES . In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES . High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL . Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate . Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

Vet Q, 2001 Jan, 23(1), 26 - 31
Review: reticuloruminal motility--a pharmacological target with a difference?
Leek BF.
The vagal sensory inputs to and motor outputs from the hindbrain gastric centres required for reticuloruminal motility were sampled directly in anaesthetized sheep using electrophysiological 'single fibre' techniques and indirectly in conscious, surgically prepared sheep . Drugs were administered by close-arterial injection into a carotid artery to observe central effects and into the coeliac artery to observe peripheral effects on the reticulorumen . Escherichia coli lipopolysaccharide produced intermediates responsible for the smooth muscle relaxation in the first phase of reticuloruminal stasis and for gastric centre depression in the second phase . Adrenergic influences on reticuloruminal motility comprise (a) an alpha1 adrenoreceptor-induced contracture and raised tension receptor sensitivity, (b) an alpha2 adrenoreceptor-mediated depression of the gastric centres causing stasis, excitation of epithelial receptors evoking rumination, and interference with acetylcholine release in the parasympathetic pathway, (c) abeta1 adrenoreceptor-mediated inhibition of the gastric centres, and (d) abeta2 adrenoreceptor-mediated inhibition of intrinsic and extrinsic motility.

Acta Physiol Hung, 2000, 87(2), 161 - 6
The effect of the glucocorticoid Oradexon on endotoxin-induced peritoneal cell response; Szakacs J et al.; Glucocorticoids are important modulators of immune reactions . They are capable of antagonising several effects of the bacterial endotoxin by inhibiting endotoxin-induced leukocyte activation, and the production of cytokines and inflammatory mediators . We earlier demonstrated that the antiglucocorticoid RU 38486 enhances the cytokine production induced by endotoxin and aggravates the course of experimental endotoxic and septic shock . In the present study we investigated the effect of the glucocorticoid Oradexon on the endotoxin-induced peritoneal cell response . For measurement of the peritoneal cell response, male CFLP mice (20-25 g) were injected i.p . with 10 microg/10 g body weight endotoxin (E . coli 026:B6 LPS, Difco Lab, Detroit, lot 110273JB) . Dexamethasone (Oradexon, N.V, Organon Oss, The Netherlands) was administered i.p., i.v . or s.c . in a dose of 0.1 mg/10 g body weight, alone or concomitantly with endotoxin . We found that bacterial endotoxin increased the total cell count due to neutrophilia at 24 hours and, due to increases in the number of macrophages and lymphocytes 48 and 72 hours after treatment, respectively . The i.p., i.v., and s.c . injection of Oradexon, increased the total cell count and the macrophage count at 24, 48 and 72 hours . The i.p., s.c . and i.v . injection of Oradexon, concomitantly with endotoxin, reduced the total cell count at 48 and 72 hours, due to decreases in the macrophage count . The i.p., i.v . or s.c . administration of Oradexon concomitantly with LPS decreased the lymphocyte count and the neutrophil count at 24 and 72 hours . These results prove that glucocorticoids are capable of modifying the immune cell reactions induced by endotoxin.

Anticancer Res, 2000 Nov-Dec, 20(6B), 4599 - 604
Different transendothelial migration behaviour pattern of blood monocytes derived from patients with benign and malignant diseases of the breast; Gebhard B et al.; BACKGROUND: In this study we compared the expression of selected monocyte surface antigens with the potential to transmigrate through an endothelial layer before and after surgery from breast cancer patients (CA) and patients with benign disease of the breast (BE) . MATERIALS AND METHODS: Transmigration capacity of mononuclear cells was determined after isolation by Ficoll density gradient, layered over human umbilical vein endothelial cells and cultured in a two chamber plate added with fMLP as a chemotactic stimulus . We determined monocyte phenotye (HLA-DR, FcgRI/CD64, CR1/CD11b and LFA-1/CD11a) and the phagocytosis of E . coli by flow cytometry . RESULTS: Before surgery blood monocytes had an equal expression of the measured surface antigens, but were different in regard to their interaction with endothelial cells . Monocytes derived from CA had a higher transmigration potency than those of BE . Moreover, the migration through the endothelial cell layer created different populations of monocytes . Surgical stress modified transmigrated monocytes of BE into the direction of monocytes from CA . Phagocytic capacity of peripheral blood monocytes from CA was significantly diminished and was further reduced after surgery when measured in transmigrated cells . CONCLUSION: Our study shows that monocytes from CA and BE can be discriminated in regard to their interaction with endothelial cells.

Arch Virol, 2000, 145(12), 2493 - 502
Mutational analysis of the NIa protease from pepper vein banding potyvirus; Joseph J et al.; The nuclear inclusion protein a (NIa) protease plays an important role in the life cycle of potyviruses by processing the viral polyprotein into functional proteins . For functional characterization, the NIa protease from Pepper vein banding potyvir s (PVBV) was overexpressed in Escherichia coli and purified . Using a recombinant polyprotein substrate containing the nuclear inclusion protein b (NIb)-coat protein (CP) cleavage site, a trans-cleavage assay was developed for the NIa protease . The polyprotein substrate also possessed the cleavage site between NIa and NIb, in addition to the NIb-CP site . However, no trans-cleavage by the NIa protease between NIa and NIb was detected indicating that the cleavage between NIa and NIb under natural conditions would be by a cis-cleavage reaction . Site-specific mutations of the conserved residues D81, D90, C110, T146, C151 and H167 were performed to investigate their roles in the catalytic process of the protease . Such an analysis has revealed that D81 and C151 constitute two of the catalytic triad residues in the NIa protease, D90 and C110 are not essential for catalysis, and T146 and H167 are probably involved in binding to Gln at the P1 position of the substrate.

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