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| Shock, 2000 Oct, 14(4), 441 - 6 Central versus peripheral mediation of naloxone's perfusion effects in endotoxic rats; Sharma AC et al.; Opioid receptor antagonists can act centrally and peripherally . It is unclear if these 2 pathways differentially mediate the perfusion-associated effects of opioid antagonism during endotoxemia . Male, Sprague-Dawley rats (340-390 g) were surgically prepared with left ventricular, tail artery, and jugular vein catheters 24 h before experiments were begun . Conscious, unrestrained rats were challenged with Escherichia coli lipopolysaccharide (LPS; 2 mg/kg/hr over 30 min) infusion . Measurements of regional blood flows were made using radioactive microspheres prior to (baseline), and at 60 and 120 min after LPS infusion . Saline (1 mL/kg bolus + 0.5 mL/kg/h infusion), naloxone (Nlx; 4 mg/kg bolus + 2 mg/kg/h infusion), or naloxone methyl bromide (Nlx-mb; 4.64 mg/kg, bolus + 2.32 mg/kg/h infusion) were administered 40 min after LPS infusion was begun . Nlx-mb does not cross the blood-brain barrier, and was thus used to differentiate central from peripherally mediated responses . At the end of each experiment, blood samples were collected for determination of ET-1 and nitric oxide metabolites (NOx = NO3 + NO2) using enzyme-linked immunosorbent assay (ELISA) and Griess reaction methods, respectively . Endotoxemia produced a significant decrease in cardiac output and an increase in systemic vascular resistance . Treatment with Nlx or Nlx-mb significantly attenuated the endotoxin-induced elevation in systemic vascular resistance and the decrease in cardiac output at 60 min after induction of endotoxemia compared with their respective baseline values . Nlx and Nlx-mb also attenuated the endotoxin-induced increases in hepatic portal and skeletal vascular resistances . These observations suggested that the ameliorative effect of Nlx on endotoxemia-induced regional vascular resistance alterations was mediated via peripheral opioid receptor mechanisms . However, although Nlx attenuated the endotoxin-induced decreases in the blood flow to the stomach and pancreas, Nlx-mb attenuated the endotoxin-induced decreases in the blood flow to the small intestine and cecum, in addition to the pancreas and, to some extent, the stomach . As such, separate central and peripherally mediated actions of opioid receptor antagonism were indicated . Nlx also resulted in an increase in the plasma levels of ET-1 only, whereas Nlx-mb increased the plasma levels of ET-1 and NOx . These observations suggest that separate central and peripheral effects of opioids during endotoxemia play a role in the associated circulatory alterations, and may differentially affect the release and/or synthesis of vasoactive mediators that might be related to their varied hepatosplanchnic vascular response during endotoxemia. Nature, 2000 Oct 12, 407(6805), 736 - 9 The population genetics of ecological specialization in evolving Escherichia coli populations; Cooper VS et al.; When organisms adapt genetically to one environment, they may lose fitness in other environments . Two distinct population genetic processes can produce ecological specialization-mutation accumulation and antagonistic pleiotropy . In mutation accumulation, mutations become fixed by genetic drift in genes that are not maintained by selection; adaptation to one environment and loss of adaptation to another are caused by different mutations . Antagonistic pleiotropy arises from trade-offs, such that the same mutations that are beneficial in one environment are detrimental in another . In general, it is difficult to distinguish between these processes . We analysed the decay of unused catabolic functions in 12 lines of Escherichia coli propagated on glucose for 20,000 generations . During that time, several lines evolved high mutation rates . If mutation accumulation is important, their unused functions should decay more than the other lines, but no significant difference was observed . Moreover, most catabolic losses occurred early in the experiment when beneficial mutations were being rapidly fixed, a pattern predicted by antagonistic pleiotropy . Thus, antagonistic pleiotropy appears more important than mutation accumulation for the decay of unused catabolic functions in these populations. Nature, 2000 Oct 12, 407(6805), 711 - 7 The crystal structure of DNA mismatch repair protein MutS binding to a G x T mismatch; Lamers MH et al.; DNA mismatch repair ensures genomic integrity on DNA replication . Recognition of a DNA mismatch by a dimeric MutS protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial . Here we present the crystal structure at 2.2 A of MutS from Escherichia coli bound to a G x T mismatch . The two MutS monomers have different conformations and form a heterodimer at the structural level . Only one monomer recognizes the mismatch specifically and has ADP bound . Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA . Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure . The interleaved nucleotide-binding sites are located far from the DNA . Mutations in human MutS alpha (MSH2/MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure. Nature, 2000 Oct 12, 407(6805), 703 - 10 Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA; Obmolova G et al.; DNA mismatch repair is critical for increasing replication fidelity in organisms ranging from bacteria to humans . MutS protein, a member of the ABC ATPase superfamily, recognizes mispaired and unpaired bases in duplex DNA and initiates mismatch repair . Mutations in human MutS genes cause a predisposition to hereditary nonpolyposis colorectal cancer as well as sporadic tumours . Here we report the crystal structures of a MutS protein and a complex of MutS with a heteroduplex DNA containing an unpaired base . The structures reveal the general architecture of members of the MutS family, an induced-fit mechanism of recognition between four domains of a MutS dimer and a heteroduplex kinked at the mismatch, a composite ATPase active site composed of residues from both MutS subunits, and a transmitter region connecting the mismatch-binding and ATPase domains . The crystal structures also provide a molecular framework for understanding hereditary nonpolyposis colorectal cancer mutations and for postulating testable roles of MutS. Cell Stress Chaperones, 2000 Oct, 5(4), 347 - 58 Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins; Hennessy F et al.; DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain . This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ . Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted . A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established . It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain . Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70. Cell Stress Chaperones, 2000 Oct, 5(4), 337 - 46 Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins; Yokota SI et al.; Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family . We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sjogren syndrome, and mixed connective tissue disease . Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera . Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls . IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL . Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65 . Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60 . Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members. Exp Mol Med, 2000 Sep 30, 32(3), 155 - 60 Identification of Escherichia coli 8-oxoguanine endonuclease; Lee YS et al.; 7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct . Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg) . However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified . In this study, we attempted to purify and identify the enzyme . Through several purification procedures, we obtained two proteins (32 kD and 29 kD) . The larger protein co-migrated with Fpg in 12% SDS-PAGE gel . Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps . These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg. J Biol Chem, 2001 Jan 19, 276(3), 1974 - 83 Epub 2000 Oct 24. Reproducing tna operon regulation in vitro in an S-30 system . Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA; Gong F et al.; Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination . Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon . Synthesis of the leader peptide, TnaC, is essential for antitermination . BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination . In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation . We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan . Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide . Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein . Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction . Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression . Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded . In the presence of inducer, antitermination increases the synthesis of the read-through transcript . TnaC synthesis is observed in the cell-free system . However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro) . Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA . The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination . 1-Methyltryptophan also is an effective inducer in vitro . This tryptophan analog is not incorporated into TnaC. J Biol Chem, 2001 Jan 19, 276(3), 1845 - 9 Epub 2000 Oct 24. Yeast glyoxalase I is a monomeric enzyme with two active sites; Frickel EM et al.; The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes . The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide . To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli . Each putative active site was inactivated by site-directed mutagenesis . According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism . The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed . Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites . The activities of the catalytic sites seem to be somewhat different . The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II) . Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction. Protein Sci, 2000 Sep, 9(9), 1791 - 800 Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides; Cai YC et al.; Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome . During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts) . Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here . Equilibrium dialysis with {3H}GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM) . Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM) . The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis . Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu . The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt) . These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP . The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex. Protein Sci, 2000 Sep, 9(9), 1709 - 18 NMR investigation of the interaction of the inhibitor protein Im9 with its partner DNase; Boetzel R et al.; The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain . Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined . For the final ensemble of 20 structures, pairwise RMSDs for residues 3-84 were 0.76 +/- 0.14 A for the backbone atoms and 1.36 +/- 0.15 A for the heavy atoms . Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 A, respectively, for residues 4-83, suggesting that binding does not cause a major conformational change in Im9 . The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H-1H-13C NOESY-HSQC spectra with and without 13C decoupling . The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures . Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping . A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed. Protein Sci, 2000 Sep, 9(9), 1685 - 99 High resolution refinement of beta-galactosidase in a new crystal form reveals multiple metal-binding sites and provides a structural basis for alpha-complementation; Juers DH et al.; The unrefined fold of Escherichia coli beta-galactosidase based on a monoclinic crystal form with four independent tetramers has been reported previously . Here, we describe a new, orthorhombic form with one tetramer per asymmetric unit that has permitted refinement of the structure at 1.7 A resolution . This high-resolution analysis has confirmed the original description of the structure and revealed new details . An essential magnesium ion, identified at the active site in the monoclinic crystals, is also seen in the orthorhombic form . Additional putative magnesium binding sites are also seen . Sodium ions are also known to affect catalysis, and five putative binding sites have been identified, one close to the active site . In a crevice on the protein surface, five linked five-membered solvent rings form a partial clathrate-like structure . Some other unusual aspects of the structure include seven apparent cis-peptide bonds, four of which are proline, and several internal salt-bridge networks . Deep solvent-filled channels and tunnels extend across the surface of the molecule and pass through the center of the tetramer . Because of these departures from a compact globular shape, the molecule is not well characterized by prior empirical relationships between the mass and surface area of proteins . The 50 or so residues at the amino terminus have a largely extended conformation and mostly lie across the surface of the protein . At the same time, however, segment 13-21 contributes to a subunit interface, and residues 29-33 pass through a "tunnel" formed by a domain interface . Taken together, the overall arrangement provides a structural basis for the phenomenon of alpha-complementation. Chem Pharm Bull (Tokyo), 2000 Oct, 48(10), 1514 - 8 Superoxide dismutase activity of iron(II)TPEN complex and its derivatives; Tamura M et al.; Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis . Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen . Thus, SODs and their functional mimics have potential value as pharmaceuticals . We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J . Biol . Chem., 264, 9243-9249 (1989)) . Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat . In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes . Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics . In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J . Organometal . Chem., in press) . Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity . Therefore our findings should be significant for the development of clinically useful SOD mimics. Hum Gene Ther, 2000 Oct 10, 11(15), 2105 - 16 Efficient transformation of primary human amniocytes by E1 functions of Ad5: generation of new cell lines for adenoviral vector production; Schiedner G et al.; Primary human cells are relatively refractory to transformation by adenoviral E1 functions . For almost two decades, human embryonic kidney (HEK)-derived 293 cells have been the only E1-complementing cell line suitable for production of E1-deleted adenoviral vectors . More recently, new vector production cell lines have been derived from human embryonic retina (HER) cells, a cell type that is difficult to obtain . We were surprised to find that readily available primary human amniocytes are efficiently transformed by adenoviral E1 functions . We selected cell lines that allow high-titer production of recombinant adenoviral vectors . The generation of replication-competent adenovirus (RCA) during production, caused by homologous recombination between vector and cellular DNA, was excluded by designing the transforming plasmid to lack sequence overlap with current adenoviral vectors . In addition, we generated an infectious plasmid that can be used for convenient generation of first-generation adenoviral vectors in Escherichia coli and that matches the E1 complementation in the new production cell lines. Environ Mol Mutagen, 2000, 36(3), 235 - 42 Mutant frequency and molecular analysis of in vivo lacI mutations in the bone marrow of Big Blue rats treated with 7, 12-dimethylbenz{a}anthracene; Shelton SD et al.; Recently, we evaluated lacI mutations in lymphocytes and mammary tissue of Big Blue (BB) rats exposed to 7, 12-dimethylbenz{a}anthracene (DMBA) . The results on the time course of mutant induction suggested that the lacI gene may manifest a tissue-specific increase in mutant frequency (MF) . To test whether a tissue-specific increase in lacI MF is dependent on the cell proliferation rate of a tissue, we examined rapidly proliferating bone marrow cells for DMBA-induced lacI mutations . Seven-week-old female BB rats were given single doses of 0, 20, and 130 mg/kg DMBA by gavage and the lacI MFs in the bone marrow were measured over a period of 14 weeks following treatment . Bone marrow cells had a remarkably low average background MF (3.1 +/- 1.6 x 10(-6) plaque-forming units) and the DMBA-induced lacI MFs were significantly higher than control MFs for both doses and at all time points (P < 0.01) . The lacI MF in the bone marrow increased for 2 weeks and then remained relatively constant; 20 and 130 mg/kg DMBA produced 34- and 106-fold increases in MF over control MF . DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base-pair substitutions and that A:T --> T:A (48%) and G:C --> T:A (24%) transversions were the predominant types . Thus, the different lacI mutation fixation times observed for bone marrow (2 weeks), mammary (10 weeks), and lymphocytes (6 weeks) suggest that the lacI gene manifests a tissue-specific mutation fixation time, which may depend on the cell proliferation rate of a tissue . In addition, the relatively low spontaneous MF in bone marrow compared with that in other tissues may be useful for increasing the sensitivity of the assay for detecting induced MFs in BB rats. J Microbiol Methods, 2000 Nov, 42(3), 233 - 44 Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters; Van Poucke SO et al.; The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization . The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete . As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E . coli/TC on a membrane filter has been developed . Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate . This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI . Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E . coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate . In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells. J Biol Chem, 2000 Dec 22, 275(51), 39811 - 4 A novel membrane anchor function for the N-terminal amphipathic sequence of the signal-transducing protein IIAGlucose of the Escherichia coli phosphotransferase system; Wang G et al.; Enzyme IIA(Glucose) (IIA(Glc)) is a signal-transducing protein in the phosphotransferase system of Escherichia coli . Structural studies of free IIA(Glc) and the HPr-IIA(Glc) complex have shown that IIA(Glc) comprises a globular beta-sheet sandwich core (residues 19-168) and a disordered N-terminal tail (residues 1-18) . Although the presence of the N-terminal tail is not required for IIA(Glc) to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIA(Glc) to the membrane-bound IIBC(Glc) . The sequence of the N-terminal tail suggests that it has the potential to form an amphipathic helix . Using CD, we demonstrate that a peptide, corresponding to the N-terminal 18 residues of IIA(Glc), adopts a helical conformation in the presence of either the anionic lipid phosphatidylglycerol or a mixture of anionic E . coli lipids phosphatidylglycerol (25%) and phosphatidylethanolamine (75%) . The peptide, however, is in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide . In addition, we show that intact IIA(Glc) also interacts with anionic lipids, resulting in an increase in helicity, which can be directly attributed to the N-terminal segment . From these data we propose that IIA(Glc) comprises two functional domains: a folded domain containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBC(Glc); and the N-terminal tail, which interacts with the negatively charged E . coli membrane, thereby stabilizing the complex of IIA(Glc) with IIBC(Glc) . This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway. J Virol, 2000 Nov, 74(22), 10846 - 51 ATP binding and ATPase activities associated with recombinant rabbit hemorrhagic disease virus 2C-like polypeptide; Marin MS et al.; The carboxy-terminal region of the rabbit hemorrhagic disease virus p37 polyprotein cleavage product has been expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein . The recombinant GST-Delta2C protein showed in vitro ATP-binding and ATPase activities . Site-directed mutagenesis studies of the conserved residues G(522) and T(529) in motif A, D(566) and E(567) in motif B, and K(600) in motif C were also performed . These results provide the first experimental characterization of a 2C-like ATPase activity in a member of the Caliciviridae. Int J Med Microbiol, 2000 Mar, 290(1), 75 - 84 Influence of pathogenicity islands and the minor leuX-encoded tRNA5Leu on the proteome pattern of the uropathogenic Escherichia coli strain 536; Piechaczek K et al.; The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries four distinct DNA regions in its chromosome, termed pathogenicity islands (PAIs I536 to IV536) . Each of these PAIs encodes at least one virulence factor . All four PAIs are associated with tRNA genes . PAI I536 and PAI II536 can be spontaneously deleted from the chromosome by homologous recombination between flanking direct repeats . The deletion of PAI II536 results in the truncation of the associated gene leuX encoding the tRNALeu . This tRNA influences the expression of various virulence traits . In order to get a deeper insight into the role of PAI I536/II536 and of the tRNA5LeU for the protein expression, the protein expression patterns of Escherichia coli 536 and different derivatives were studied . Differences in the protein expression patterns of the wild-type strain Escherichia coli 536, its mutants 536-21 (PAI I536-, PAI II536-, leuX-), 536delta102 (PAI I536+, PAI II536+, leuX-) as well as of the strain 536R3 (PAI I536-, PAI II536-, leuX+) were analyzed by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry . We identified about 39 different intracellular proteins whose expression is markedly altered in the different strain backgrounds . These differences can be linked either to the presence or absence of the PAI I536 and PAI II536 or to that of the tRNA gene leuX . The identities of 34 proteins have been determined by MALDI-TOF-MS . The identification of five proteins was not possible . The results suggest that proteome analysis is an efficient approach to study differences in global gene expression . The comparison of protein expression patterns of the uropathogenic E . coli strain 536 and different derivatives revealed that in this strain the expression of various proteins including those encoded by many housekeeping genes is affected by the presence of PAI I536 and Pai II536 or by that of the tRNA5Leu. Arch Virol, 2000, 145(9), 1895 - 908 Indian citrus ringspot virus: a proposed new species with some affinities to potex-, carla-, fovea- and allexiviruses; Rustici G et al.; An isolate of Indian citrus ringspot virus from Kinnow mandarin in northern India had flexuous particles with evident cross-banding and a modal length of 650 nm . It was mechanically transmitted to five herbaceous hosts including Phaseolus vulgaris cv Saxa, in which it became systemic . In thin sections, virus particles were observed in the cytoplasm of parenchyma cells but no specific inclusions were seen . The virus was purified from infected Saxa bean leaves and an antiserum prepared . There was no serological cross-reaction with representative allexi-, capillo-, potex- and trichoviruses, except a faint one-way reaction with Potato virus X . Purified virus yielded a major band, the presumed coat protein (CP), of about 34 kDa, and a single ssRNA of about 7.5 kb, which was infectious . Two ORFs encoding putative proteins of 34 kDa and 23 kDa were located in the 3' part of the RNA . The product of the 34 kDa ORF was confirmed as the CP by expression in E . coli . The derived amino acid sequence of the CP contained some short motifs similar to those of potex-, fovea-, carla- and allexiviruses but otherwise there was no strong similarity to any of these . The 23 kDa ORF contained a zinc finger-like sequence, as in similar ORFs in carla- and allexiviruses but overall amino acid homology with these was low . The virus does not appear to fall into any known genus . A new species is proposed . Serological and molecular diagnostic reagents were prepared. J Protein Chem, 2000 May, 19(4), 319 - 26 Probing the roles of the only universally conserved leucine residue (Leu122) in the oligomerization and chaperone-like activity of Mycobacterium tuberculosis small heat shock protein Hsp16.3; Dai H et al.; To understand the role of the only universally conserved hydrophobic residue among all the members of the sHsp family, this extremely well conserved Leu122 residue in Hsp16.3 was replaced by valine, alanine, asparigine, or aspartate residues . Only very small amounts of the L122D and L122N mutant Hsp16.3 proteins were expressed in the transformed E . coli; however, both the L122V and L122A were readily expressed . The L122V and L122A mutant proteins had similar oligomeric structures to the wild-type protein at room temperature . Examination of the L122A mutant protein by native pore gradient PAGE and CD spectroscopy, however, revealed a smaller oligomeric size and different secondary structure at 37 degrees C . Both L122V and L122A mutant proteins exhibited significantly lowered chaperone activities . Observations reported here suggest a very important role of this only universally conserved Leu residue in both the formation of specific oligomeric structures and the molecular chaperone activities of Hsp16.3. Mol Endocrinol, 2000 Oct, 14(10), 1536 - 49 The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation; Martini JF et al.; We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death . Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration . The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B . 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity . Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD) . Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis . These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells. Cell Death Differ, 2000 Sep, 7(9), 785 - 94 CD40 ligand, Bcl-2, and Bcl-xL spare group I Burkitt lymphoma cells from CD77-directed killing via Verotoxin-1 B chain but fail to protect against the holotoxin; Gordon J et al.; Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL) . The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack . Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand . Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain . In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin . These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed. Biochemistry (Mosc), 2000 Sep, 65(9), 1097 - 104 The major phospholipid of Escherichia coli, phosphatidylethanolamine, is required for efficient production and secretion of alkaline phosphatase; Golovastov VV et al.; The major phospholipid of the Escherichia coli membranes--the zwitterion phosphatidylethanolamine (PE)--is the only phospholipid involved in the formation of non-bilayer structure of membrane lipids, which is supposed to be necessary for efficient translocation of secreted proteins across the cytoplasmic membrane . The effect of PE on the production and secretion of alkaline phosphatase has been studied in this work using the mutant strain E . coli AD93, which is unable to synthesize PE . It was shown that this phospholipid is required for the efficient production and secretion of alkaline phosphatase . The anionic phospholipid cardiolipin in combination with divalent cations Mg2+ functionally replaces PE in these processes, participating in the regulation of lipid polymorphism. Biochemistry (Mosc), 2000 Sep, 65(9), 1075 - 81 The primary structure of the N-terminal region of mature alkaline phosphatase is critical for secretion and function of the enzyme; Kononova SV et al.; The export signal has been assumed to be localized not only in the signal peptide of a secreted protein precursor, but also in the N-terminal region of the mature polypeptide chain . Mutant alkaline phosphatases with amino acid substitutions of two positively charged residues (Lys or Arg) in this region at different distances from the signal peptide have been studied to test this assumption . The efficiency of secretion has been shown to decrease in mutant proteins with amino acid substitutions in the region of 16-18 amino acid residues; the closer to the signal peptide is the substitution, the greater is the decrease . A change in the primary structure of the N-terminal domain results also in an increase in the Michaelis constant, which is greater the farther is the amino acid substitution from the signal peptide, suggesting a change in the enzyme function as well. Biochemistry (Mosc), 2000 Sep, 65(9), 1011 - 8 Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11; Dubnovitsky AP et al.; Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies) . The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC) . Subsequent renaturation results in approximately 99% pure preparation with high yield . The VL-domain forms dimers at concentrations from 1 to 10 mg/ml . Monomeric form is detected only at protein concentrations below 0.5 mg/ml . Functional activity of the VL-domain was verified by two variants of ELISA . The affinity of the VL-domain ((0.2-1.2) . 108 M(-1)) is similar to the affinity of the full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-fold lower than that in the case of F11 antibody . In another ELISA system with immobilized ferritin, affinity was decreased 30-fold . The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain . The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins. Biochemistry (Mosc), 2000 Sep, 65(9), 1006 - 10 A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis; Sergeev VN et al.; Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease . Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed . When expressed in E . coli, all these plasmids displayed EcoRII endonuclease activity . We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein . This mutant protein had no EcoRII endonuclease activity . The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site . However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease. FEBS Lett, 2000 Oct 20, 483(2-3), 181 - 5 Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase; Balzarini J et al.; A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase) . The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase . These inhibitors reversibly inhibited TPase . Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate . In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate . Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme . To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors. FEBS Lett, 2000 Oct 20, 483(2-3), 165 - 8 Identification of triphosphoribosyl-dephospho-CoA as precursor of the citrate lyase prosthetic group; Schneider K et al.; The gamma-subunit of citrate lyase (EC 4.1.3.6) contains the prosthetic group 2'-(5"-phosphoribosyl)-3'-dephospho-CoA and serves as an acyl carrier protein (ACP) . We recently showed that in Escherichia coli the proteins CitG and CitX are essential for holo-ACP synthesis and provided evidence that CitG catalyzes the formation of a prosthetic group precursor from ATP and dephospho-CoA, which is subsequently attached via phosphodiester linkage to apo-ACP by CitX . Here we prove that CitG indeed catalyzes the conversion of ATP and dephospho-CoA to adenine and 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA, the predicted precursor of the prosthetic group . Furthermore, this precursor was transferred by CitX to apo-ACP, yielding holo-ACP . Thus, our proposed mechanism for holo-ACP synthesis could be verified. J Biol Chem, 2001 Jan 12, 276(2), 1204 - 10 Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase; Panisko EA et al.; Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2 . IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP . In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods . A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme . A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites . Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2 . We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo . Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1 . A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate . A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP . Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other. J Biol Chem, 2000 Dec 15, 275(50), 38961 - 4 Selective role of G protein gamma subunits in receptor interaction; Hou Y et al.; Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (alphabetagamma) is the primary event-modulating signaling by G proteins . The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the betagamma complex, in receptor activation are unclear . In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers alphai2beta1gamma5 and alphai2beta1gamma7 with equal efficacy . However, when the alpha subunit type is substituted with alphao, alphaobeta1gamma7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with alphaobeta1gamma5 . Using a sensitive assay based on betagamma complex stimulation of phospholipase C activity, we show that both beta1gamma5 and beta1gamma7 form heterotrimers equally well with alphao and alphai . These results indicate that the gamma subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer. Biochem J, 2000 Nov 1, 351 Pt 3, 717 - 22 Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins; Sharma DK et al.; We previously established an in vitro assay for glycosylphosphatidylinositol (GPI) anchoring of proteins using trypanosome membranes . We now show that GPI anchoring is lost when the membranes are washed at high pH and restored to physiological pH prior to assay . We show that soluble component(s) of the endoplasmic reticulum that are lost in the high-pH wash are required for GPI anchoring . We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity . Size fractionation of the high-pH extract indicated that the active component(s) was 30-50 kDa in size and was inactivated by iodoacetamide . Activity could also be restored by reconstituting the inactivated membranes with Escherichia coli-expressed, polyhistidine-tagged Leishmania mexicana GPI8 (GPI8-His; L . mexicana GPI8 is a soluble homologue of yeast and mammalian Gpi8p) . No activity was seen when iodoacetamide-treated GPI8-His was used; however, GPI8-His could restore activity to iodoacetamide-treated membranes . Antibodies raised against L . mexicana GPI8 detected a protein of approx . 38 kDa in an immunoblot of the high-pH extract of trypanosome membranes . Our data indicate (1) that trypanosome GPI8 is a soluble lumenal protein, (2) that the interaction between GPI8 and other putative components of the transamidase may be dynamic, and (3) that GPI anchoring can be biochemically reconstituted using an isolated transamidase component. Biochem J, 2000 Nov 1, 351 Pt 3, 551 - 5 Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P(4) 1-kinase/Ins(1,3,4)P(3) 5/6-kinase; Yang X et al.; We describe a human cDNA encoding 1-kinase activity that inactivates Ins(3,4,5,6)P(4), an inhibitor of chloride-channel conductance that regulates epithelial salt and fluid secretion, as well as membrane excitability . Unexpectedly, we further discovered that this enzyme has alternative positional specificity (5/6-kinase activity) towards a different substrate, namely Ins(1,3,4)P(3) . Kinetic data from a recombinant enzyme indicate that Ins(1,3,4)P(3) (K(m)=0.3 microM; V(max)=320 pmol/min per microg) and Ins(3,4,5,6)P(4) (K(m)=0.1 microM; V(max)=780 pmol/min per microg) actively compete for phosphorylation in vivo . This competition empowers the kinase with multitasking capability in several key aspects of inositol phosphate signalling. J Struct Biol, 2000 Aug, 131(2), 164 - 9 Preliminary X-ray crystallographic and NMR studies on the exonuclease domain of the epsilon subunit of Escherichia coli DNA polymerase III; Hamdan S et al.; The structured core of the N-terminal 3'-5' exonuclease domain of epsilon, the proofreading subunit of Escherichia coli DNA polymerase III, was defined by multidimensional NMR experiments with uniformly (15)N-labeled protein: it comprises residues between Ile-4 and Gln-181 . A 185-residue fragment, termed epsilon(1-185), was crystallized by the hanging drop vapor diffusion method in the presence of thymidine-5'-monophosphate, a product inhibitor, and Mn(2+) at pH 5.8 . The crystals are tetragonal, with typical dimensions 0.2 mm x 0.2 mm x 1.0 mm, grow over about 2 weeks at 4 degrees C, and diffract X-rays to 2.0 A . The space group was determined to be P4(n)2(1)2 (n = 0, 1, 2, 3), with unit cell dimensions a = 60.8 A, c = 111.4 A . J Struct Biol, 2000 Aug, 131(2), 96 - 107 Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta8 outer membrane protein Omp21 from Comamonas acidovorans; Baldermann C et al.; The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel . This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other related molecules . After we established an expression system, recombinant Omp21 was purified by Ni(2+) chelation affinity chromatography and refolded in situ while bound to resin . The native state of refolded protein was proven by FTIR spectroscopy and monitored with denaturing PAGE (heat modification) . Both native and recombinant Omp21 were reconstituted in lipid membranes and crystallized two-dimensionally by controlled dialysis . Recombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees . The 3-D structure of negatively stained, recombinant Omp21 was determined at a resolution of 1.8 nm by means of electron crystallography . Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity between the mass distribution of exoplasmic loops of Omp21 and OmpA . Growth Horm IGF Res, 2000 Oct, 10(5), 248 - 55 A novel bioassay based on human growth hormone (hGH) receptor mediated cell proliferation: measurement of 20K-hGH and its modified forms; Ikeda M et al.; Previously we introduced the full-length hGH receptor (hGHR) into the mouse pro-B cell line, Ba/F3, and obtained stable transfectant (Ba/F3-hGHR), which could grow in response to 20K- and 22K-hGH in a dose-dependent manner(1) . In the present study, we established a new bioassay system based on the proliferation of the Ba/F3-hGHR in combination with the eluted stain assay (ESTA) . The Ba/F3-hGHR assay is completed in 18 h and requires only 10(-6)-fold amount of GH sample (1.8 ng) as compared with the rat weight gain assay . The validation study shows that the Ba/F3-hGHR assay is specific for hGH, precise (RSD = 1.1-19.7%) and ultrasensitive (lower limit of working range = 18.7 pg/mL) . Four modified forms of recombinant 20K-hGH (oxidized, deamidated, des-Phe(1)and cleaved form) all of which are newly identified were measured by the Ba/F3-hGHR assay and the rat weight gain assay with our in-house recombinant 20K-hGH as standard . The oxidized and deamidated 20K-hGH were fully active, however the des-Phe(1)and cleaved 20K-hGH had significantly reduced activities in both assays . These findings suggest that the Ba/F3-hGHR assay is useful as an alternative to the rat weight gain assay . J Biol Chem, 2001 Jan 26, 276(4), 2808 - 15 Epub 2000 Oct 20. Mutations that affect ligand binding to the Escherichia coli aspartate receptor: implications for transmembrane signaling; Bjorkman AM et al.; Three arginine residues of the binding site of the Escherichia coli aspartate receptor contribute to its high affinity for aspartate (K(d) approximately 3 microm) . Site-directed mutations at residue 64 had the greatest effect on aspartate binding . No residue could substitute for the native arginine; all changes resulted in an apparent K(d) of approximately 35 mm . These mutations had little impact on maltose responses . At residue Arg-69, a lysine substitution was least disruptive, conferring an apparent K(d) of 0.3 mm for aspartate . Results obtained for an alanine mutant were similar to those with cysteine and histidine mutants (K(d) approximately 5 mm) indicating that side chain size was not an important factor here . Proline and aspartate caused more severe defects, presumably for reasons related to conformation and charge . The impact of residue 69 mutations on the maltose response was small . Mutations at Arg-73 had similar effects on aspartate binding (K(d) 0.3-7 mm) but more severe consequences for maltose responses . Larger side chains resulted in the best aspartate binding, implying steric considerations are important here . Signaling in the mutant proteins was surprisingly robust . Given aspartate binding, signaling occurred with essentially wild-type efficiency . These results were evaluated in the context of available structural data. J Biol Chem, 2001 Jan 19, 276(3), 1837 - 44 Epub 2000 Oct 20. In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase; Leimkuhler S et al.; We were able to reconstitute molybdopterin (MPT)-free sulfite oxidase in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate . MPT-free human sulfite oxidase apoprotein was obtained by heterologous expression in an Escherichia coli mutant with a defect in the early steps of MPT biosynthesis . In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor Z, purified MPT synthase, and sodium molybdate . In vitro synthesized MPT generated from precursor Z by MPT synthase remains bound to the synthase . Surprisingly, MPT synthase was found capable of donating bound MPT to MPT-free sulfite oxidase . MPT was not released from MPT synthase when either bovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase . After the inclusion of sodium molybdate in the reconstitution mixture, active sulfite oxidase was obtained, revealing that in vitro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presence of high concentrations of molybdate . The conversion of MPT into Moco by molybdate chelation apparently occurs concomitantly with the insertion of MPT into sulfite oxidase. J Biol Chem, 2001 Feb 2, 276(5), 3037 - 45 Epub 2000 Oct 19. Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains; Polyak SW et al.; Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes . The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate . We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL . Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C . The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature . This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain . In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay . Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect . Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat) . The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide . Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL. Plant Cell, 2000 Oct, 12(10), 1951 - 60 Insertion of OEP14 into the outer envelope membrane is mediated by proteinaceous components of chloroplasts; Tu SL et al.; Most chloroplastic outer envelope membrane proteins are synthesized in the cytosol at their mature size without a cleavable targeting signal . Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP . The insertion has been assumed to be mediated by a spontaneous mechanism or by interaction solely with the lipid components of the outer membrane . However, we show here that insertion of an outer membrane protein requires some trypsin-sensitive and some N-ethylmaleimide-sensitive components of chloroplasts . Association and insertion of the outer membrane protein are saturable and compete with the import of another outer membrane protein . These data suggest that import of chloroplastic outer membrane proteins occurs at specific proteinaceous sites on chloroplasts. Plant Cell, 2000 Oct, 12(10), 1903 - 16 AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region; Morisawa G et al.; Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions . We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs . A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook . For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo . AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm . AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus . AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs . J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. Biochemistry, 2000 Oct 24, 39(42), 12959 - 69 Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein; Galletto R et al.; Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques . This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding . The stoichiometry of the DnaC-nucleotide complex has been determined in direct binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP . The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT) . The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer . Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution . Although the presence of the gamma-phosphate does not affect the affinity, the structure of the triphosphate group is critical . While the affinity of ATP-gamma-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are approximately 2 and approximately 4 orders lower than that of ATP, respectively . Moreover, the ribose plays a significant role in forming a stable complex . The binding constants of dATP and dADP are approximately 2 orders of magnitude lower than those for ribose nucleotides . The nucleotide-binding site of the DnaC protein is highly base specific . The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides . The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity . The significance of the results for the functioning of the DnaC protein is discussed. Biochemistry, 2000 Oct 24, 39(42), 12853 - 61 Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding; Onesti S et al.; Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys) . The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate . A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding . The lysine substrate is involved in a network of hydrogen bonds . Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees . The result of these changes is a closing up of the active site upon lysine binding. Biochemistry, 2000 Oct 24, 39(42), 12789 - 95 High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase; Kitahara R et al.; A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar . Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first . Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first . The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units . The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10% . The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar . These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function. Biochemistry, 2000 Oct 24, 39(42), 12747 - 52 Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy; Uchida T et al.; The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy . On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO {Uchida et al . (1998) J . Biol . Chem . 273, 19988-19992}, whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported {Vogel et al . (1999) Biochemistry 38, 2679-2687} . Although our previous resonance Raman study failed to detect the Fe-His stretching {nu(Fe-His)} mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to nu(Fe-His) at 211 cm(-)(1) immediately after photolysis, while it became nondiscernible after 100-ps delay . The low nu(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA . This and the rapid recombination of CO characterize the heme pocket of CooA . The 211 cm(-)(1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im) . Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator. Can J Vet Res, 2000 Oct, 64(4), 208 - 11 Yohimbine ameliorates the effects of endotoxin on gastric emptying of the liquid marker acetaminophen in horses; Meisler SD et al.; The effect of yohimbine pretreatment on gastric emptying of a liquid marker in horses was evaluated by measuring serum concentrations of acetaminophen . Gastric emptying was determined in normal, fasted horses, in horses given endotoxin (E . coli 055 B5; 0.2 microg/kg) intravenously, and in horses given yohimbine (0.25 mg/kg, IV, over 30 minutes) plus endotoxin . Acetaminophen (20 mg/kg) was given by stomach tube 15 minutes after the endotoxin infusion . Blood samples for acetaminophen analysis were collected, and time to reach the peak serum concentration (Tmax), the maximum serum concentration (Cmax) and the area under the acetaminophen serum concentration versus time curve (AUC) were determined for each treatment group . Endotoxin significantly increased Tmax, indicating a profound delay in gastric emptying and yohimbine pretreatment significantly (P < or = 0.05) prevented this effect. Arch Microbiol, 2000 Sep, 174(3), 168 - 74 Seasonal and spatial community dynamics in the meromictic Lake Cadagno; Bosshard PP et al.; The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments . Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8-536) and another specific for the family Chromatiaceae (E . coli positions 8-1005) . The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE . The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns . For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae . This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer . All dominant bands were excised and sequenced . Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year . In the chemocline, Chomatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter . This change was confirmed by fluorescent in situ hybridization. Biol Pharm Bull, 2000 Oct, 23(10), 1147 - 52 NMR structure of ribonuclease HI from Escherichia coli; Fujiwara M et al.; The solution structure of ribonuclease HI (RNase HI) from Escherichia coli (E . coli), a protein of 155 residues, was determined . Three-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain 1,424 distance constraints between individually assigned polypeptide chain hydrogen atoms . Supplemental geometric constraints of 90phi angles and 12chi1 angles, and the distance constraints of 66 hydrogen bonds were experimentally derived . Using the DADAS90 program that calculates structures in dihedral angle space, 15 structures satisfying almost all constraints were obtained . The average root mean square deviation (RMSD) from the mean structure was 0.75 A for backbone atoms . The RMSD for backbone atoms between the representative NMR structure with the smallest constraint violation and crystal structures was within 1.2 A . Although the NMR and crystal structures thus resemble one another, a significant discrepancy was observed in a region termed 'basic protrusion.' The discrepancy observed in NMR experiments is explained by fluctuation in this region. Bioorg Khim, 2000 Aug, 26(8), 601 - 4 {Identification of Escherichia coli nitrate reductase as an antigen for a monoclonal antibody with previously unknown specificity}; Korneenko TV et al.; The immunoaffinity chromatography of total membrane proteins from Escherichia coli helped determine the specificity of the monoclonal antibody 3A6 that was obtained upon immunization of mice with nicotinamide nucleotide transhydrogenase preparations and reacted with an unknown E . coli antigen . Proteins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified by N-terminal sequencing as the subunits of nitrate reductase . This conclusion was confirmed by immunoblotting with the 3A6 antibody of the proteins from the E . coli cells grown upon induction of nitrate reductase . It was shown that the 3A6 antibody specifically recognizes the alpha subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity. J Mol Evol, 2000 Oct, 51(4), 404 - 15 Mitochondrial DNA of Hydra attenuata (Cnidaria): a sequence that includes an end of one linear molecule and the genes for l-rRNA, tRNA(f-Met), tRNA(Trp), COII, and ATPase8; Pont-Kingdon G et al.; The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined . This segment contains complete genes for tRNA(f-Met), l-rRNA, tRNA(Trp), subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5' 136 ntp of ATPase6 . These genes are arranged in the order given and are transcribed from the same strand of the molecule . As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H . attenuata is near standard . The only modification appears to be that TGA specifies tryptophan rather than termination . Also as in M . senile and S . glaucum, the encoded H . attenuata mt-tRNA(f-Met) has primary and secondary structural features resembling those of Escherichia coli initiator tRNA(t-Met) . As the encoded mt-tRNA(Trp) cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested . The encoded H . attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M . senile mt-l-rRNA . Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H . attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements. Genes Dev, 2000 Oct 15, 14(20), 2570 - 5 The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication; Liao S et al.; The Bloom's syndrome gene (BLM) plays a pivotal role in the maintenance of genomic stability in somatic cells . It encodes a DNA helicase (BLM) of the RecQ family, but the exact function of BLM remains elusive . To study this question, we have cloned the BLM homolog of the frog Xenopus laevis (xBLM) and have raised antibodies to it . Immunodepletion of xBLM from a Xenopus egg extract severely inhibits the replication of DNA in reconstituted nuclei . Moreover, the inhibition can be rescued by the addition of the recombinant xBLM protein . These results provide the first direct evidence that BLM plays an important role in DNA replication, suggesting that Bloom's syndrome may be the consequence of defective DNA replication. Virology, 2000 Oct 25, 276(2), 445 - 54 Structure of simian virus 40 DNA replicated by herpes simplex virus type 1; Blumel J et al.; Replicating herpes simplex virus type 1 (HSV-1) DNA is known to form large branched structures . The aim of this study was to define whether HSV-1-specific DNA elements in cis play a critical role in formation of this structure . We did this by investigating the structure of heterologous simian virus 40 (SV40) DNA, which is replicated in HSV-infected cells by SV40 large T-antigen and defined HSV-encoded replication factors (e.g., DNA polymerase, single-stranded DNA-binding protein, and helicase-primase) . During this process, extrachromosomal concatemeric DNA replication products are formed, indicating a herpesvirus-specific replication mode . In this study, we found that the replicating SV40 DNA consisted of a complex branched structure indistinguishable from that of replicating HSV DNA . Thus, no HSV-specific DNA element is necessary in cis for the formation of the large branched structure during HSV DNA replication . The trans-acting HSV DNA replication proteins seem to be sufficient to generate these complex structures . Moreover, replicating SV40 DNA showed a high frequency of homologous recombination events, which is typical for HSV DNA replication . However, in contrast to HSV origin-bearing amplicon plasmids, SV40 plasmids bearing the HSV cleavage-packaging signal were not efficiently processed to linear 150-kb DNA packaged into HSV capsids . This indicates that initiation of DNA synthesis on HSV-ori determines some, yet undefined, property of replicating HSV DNA, which is crucial for regular processing of the replication intermediates to daughter genomes . Virology, 2000 Oct 25, 276(2), 376 - 87 The structural protein E of the archaeal virus phiCh1: evidence for processing in Natrialba magadii during virus maturation; Klein R et al.; phiCh1 is a lysogenic virus for the haloalkalophilic archaeon Natrialba magadii . The virus morphology resembles other members of Myoviridae infecting Halobacterium species . The gene of the major capsid protein E of virus phiCh1 was cloned and the DNA sequence was determined . Gene E was mapped to a 3.2-kbp ClaI fragment, localized to the 5'-end of the phiCh1 genome . The complete nucleotide sequence of this region was determined and the identity of gene E was confirmed by comparing the experimentally determined N-terminal amino acid sequence of the purified protein to the translated DNA sequence of its open reading frame . We present evidence that the gene E product is proteolytically cleaved between Lys(16) and Asn(17) to yield the 305 residue polypeptides found in the mature viral capsid . Processing of the protein itself during virus development was determined by 2D gel electrophoresis using protein E-specific antibodies . Sequence similarity studies revealed an 80% identity to capsid protein Hp32 of phiH, infecting Halobacterium salinarum . RT-PCR analysis as well as Western blot studies revealed gene E as a late gene . Transcripts and proteins could be detected shortly before onset of lysis of the lysogenic strain N . magadii L11 . Virology, 2000 Oct 25, 276(2), 364 - 75 Puumala (PUU) hantavirus strain differences and insertion positions in the hepatitis B virus core antigen influence B-cell immunogenicity and protective potential of core-derived particles; Koletzki D et al.; Hepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hallnas) nucleocapsid (N) protein sequence (aa 1-45), alternatively inserted at three distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hallnas) N (aa 1-45) readthrough protein were generated . Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40-50% of the animals showed markers of protection . In contrast, internal insertion of PUU strain CG18-20 N (aa 1-45) into the HBV core caused a highly protective immune response in the bank vole model . Immunizations with particles carrying aa 75-119 of PUU (CG18-20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein . A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles . Except for the exclusive occurrence of antibodies directed against aa 231-240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge . J Autoimmun, 2000 Nov, 15(3), 347 - 57 Endogenous ecotropic murine leukemia viral (MuLV) envelope protein as a new autoantigen reactive with non-obese diabetic mice sera; Choi SE et al.; The identification and characterization of autoantigens associated with autoimmune IDDM (insulin dependent diabetes mellitus) would help to elucidate the pathogenic mechanism of this disease as well as to design antigen-based immunotherapy . Non-obese diabetic (NOD) mice have been used as the best model for studying the pathogenesis of human IDDM . To identify new autoantigens associated with IDDM, the lambda gt11-cDNA library from MIN6N8a, NOD-derived pancreatic beta cell line, was constructed and then candidate autoantigen clones were screened with prediabetic NOD sera . Nine positive clones were selected from 2x10(5)phage plaques . The nucleotide sequencing and homology searching showed that six of the nine positive clones had part of the endogenous ecotropic murine leukemia viral (MuLV) envelope gene . Nested deletion of this envelope gene revealed that the leucine zipper region in the transmembrane domain of MuLV envelope protein was the target epitope(s) reactive with prediabetic NOD mice sera . The prevalence of MuLV envelope protein-positive antibody in NOD mice was around 46%, while the non-NOD mice strains including BALB/c, ICR, C57BL/6, and SJL/J mice did not produce this envelope protein-reactive antibody . The expression of endogenous ecotropic MuLV envelope gene in NOD mouse pancreas was distinct in those with severe insulitis . However, both prediabetic and diabetic NOD mice did not show the MHC class II-restrictive cellular autoimmunity against our purified recombinant envelope protein . In this study, we showed that the endogenous ecotropic MuLV envelope protein was a new autoantigen reactive with the activated NOD humoral immune system . Science, 2000 Oct 20, 290(5491), 481 - 6 Structure of a glycerol-conducting channel and the basis for its selectivity; Fu D et al.; Membrane channel proteins of the aquaporin family are highly selective for permeation of specific small molecules, with absolute exclusion of ions and charged solutes and without dissipation of the electrochemical potential across the cell membrane . We report the crystal structure of the Escherichia coli glycerol facilitator (GlpF) with its primary permeant substrate glycerol at 2.2 angstrom resolution . Glycerol molecules line up in an amphipathic channel in single file . In the narrow selectivity filter of the channel the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor, and donor atoms . Two conserved aspartic acid-proline-alanine motifs form a key interface between two gene-duplicated segments that each encode three-and-one-half membrane-spanning helices around the channel . This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded. Clin Orthop, 2000 Oct, (379 Suppl), S252 - 5 In vivo gene transfer into tendon by recombinant adenovirus; Lou J; Recombinant adenovirus mediated Escherichia coli lacZ gene transfer into chicken tendon and tendon sheath has been reported in the current study . The constructed recombinant virus carrying lacZ gene was injected between tendon and tendon sheath to conduct in vivo gene transfer . During the course of the study, each tendon received a 10 uL injection containing 10(5) plaque forming units of recombinant adenovirus with beta-galactosidase gene . The samples were harvested at 3 days, 30 days, and 75 days after injection . For the virus dose-transduction rate study, five different doses were injected to groups of chicken tendons . LacZ gene transfer was detected for its coding product beta-galactosidase by staining with X-gal solution . Results showed that the tendon and tendon sheath received the gene transfer with blue staining . The transferred lacZ gene remained stable for 75 days in the tendon and tendon sheath . A virus dose-dependent pattern of transduction rate was observed in the gene transferred tendons . The area of tendon transduction was approximately 2% for 3 x 10(6) plaque forming units recombinant adenovirus with beta-galactosidase versus 40% for 6 x 10(7) plaque forming units recombinant adenovirus with beta-galactosidase gene . The data suggested that functional exogene could be transferred into the tendon and tendon sheath by the same strategy to improve healing and avoid adhesion. J Vet Med Sci, 2000 Sep, 62(9), 941 - 5 Comparative analysis of the putative amino acid sequences of chlamydial heat shock protein 60 and Escherichia coli GroEL; Ochiai Y et al.; The nucleotide sequences of the gene encoding chlamydial heat shock protein 60 (cHSP60) of 7 Chlamydia psittaci strains were determined . Comparison of sequences of the cHSP60 gene among chlamydiae showed high identities of the nucleotide sequences by 81.0% or greater and of the deduced amino acid sequences by 92.2% or greater . Comparison of the amino acid sequences between chlamydia and the other bacterial HSP60s resulted in the finding of three highly conserved regions, suggesting that these regions play a role in some function . In addition, 26- or 27-functional residues in the Escherichia coli GroEL out of the 28-residues are conserved in the amino acid sequences of the cHSP60 . The data suggest that the function of the cHSP60 may be the same as that of the E . coli GroEL. J Biol Chem, 2001 Jan 12, 276(2), 1233 - 43 A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase; Santolini J et al.; After initiating NO synthesis a majority of neuronal NO synthase (nNOS) quickly partitions into a ferrous heme-NO complex . This down-regulates activity and increases enzyme K(m,O(2)) . To understand this process, we developed a 10-step kinetic model in which the ferric heme-NO enzyme forms as the immediate product of catalysis, and then partitions between NO dissociation versus reduction to a ferrous heme-NO complex . Rate constants used for the model were derived from recent literature or were determined here . Computer simulations of the model precisely described both pre-steady and steady-state features of nNOS catalysis, including NADPH consumption and NO production, buildup of a heme-NO complex, changes between pre-steady and steady-state rates, and the change in enzyme K(m,O(2)) in the presence or absence of NO synthesis . The model also correctly simulated the catalytic features of nNOS mutants W409F and W409Y, which are hyperactive and display less heme-NO complex formation in the steady state . Model simulations showed how the rate of heme reduction influences several features of nNOS catalysis, including populations of NO-bound versus NO-free enzyme in the steady state and the rate of NO synthesis . The simulation predicts that there is an optimum rate of heme reduction that is close to the measured rate in nNOS . Ratio between NADPH consumption and NO synthesis is also predicted to increase with faster heme reduction . Our kinetic model is an accurate and versatile tool for understanding catalytic behavior and will provide new perspectives on NOS regulation. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7667 - 7672 In vitro reconstitution of the photosystem I light-harvesting complex LHCI-730: Heterodimerization is required for antenna pigment organization; Schmid VH et al.; Here we describe the in vitro reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato Lhca genes in Escherichia coli . Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigment-proteins, whereas a combination thereof yields a dimeric complex . Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization . The reconstituted Lhca1/4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry . Monomeric complexes of Lhca1 or Lhca4 possess lower pigment/protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments . In addition to higher averages of chlorophyll a/b ratios in monomeric complexes than in LHCI-730, comparative fluorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b. Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 14199 - 14203 Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP(+) oxidoreductase; Somssich IE et al.; We expressed a cDNA encoding the Arabidopsis thaliana defense-related protein ELI3-2 in Escherichia coli to determine its biochemical function . Based on a protein database search, this protein was recently predicted to be a mannitol dehydrogenase {Williamson, J . D., Stoop, J . M . H., Massel, M . O., Conkling, M . A . & Pharr, D . M . (1995) Proc . Natl . Acad . Sci . USA 92, 7148-7152} . Studies on the substrate specificity now revealed that ELI3-2 is an aromatic alcohol: NADP(+) oxidoreductase (benzyl alcohol dehydrogenase) . The enzyme showed a strong preference for various aromatic aldehydes as opposed to the corresponding alcohols . Highest substrate affinities were observed for 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, and benzaldehyde, in this order, whereas mannitol dehydrogenase activity could not be detected . These and previous results support the notion that ELI3-2 has an important role in resistance-related aromatic acid-derived metabolism. J Biol Chem, 2001 Feb 2, 276(5), 3247 - 53 Epub 2000 Oct 18. High resolution structure of the phosphohistidine-activated form of Escherichia coli cofactor-dependent phosphoglycerate mutase; Bond CS et al.; The active conformation of the dimeric cofactor-dependent phosphoglycerate mutase (dPGM) from Escherichia coli has been elucidated by crystallographic methods to a resolution of 1.25 A (R-factor 0.121; R-free 0.168) . The active site residue His(10), central in the catalytic mechanism of dPGM, is present as a phosphohistidine with occupancy of 0.28 . The structural changes on histidine phosphorylation highlight various features that are significant in the catalytic mechanism . The C-terminal 10-residue tail, which is not observed in previous dPGM structures, is well ordered and interacts with residues implicated in substrate binding; the displacement of a loop adjacent to the active histidine brings previously overlooked residues into positions where they may directly influence catalysis . E . coli dPGM, like the mammalian dPGMs, is a dimer, whereas previous structural work has concentrated on monomeric and tetrameric yeast forms . We can now analyze the sequence differences that cause this variation of quaternary structure. J Biol Chem, 2001 Jan 19, 276(3), 2098 - 107 Epub 2000 Oct 18. Functional dissection of the LysR-type CysB transcriptional regulator . Regions important for DNA binding, inducer response, oligomerization, and positive control; Lochowska A et al.; CysB is a tetrameric LysR-type transcriptional regulator that acts as an activator of cys regulon genes and as an autorepressor . Positive control of cys genes requires the presence of the inducer N-acetylserine . Following random and site-directed mutagenesis of the cysB gene, 20 CysB variants were isolated . Six single amino acid substitutions within the N terminus of CysB abolished the DNA-binding ability of the protein . Seven mutations in the central region of CysB affected its response to the inducer . Four of these CysB mutants retained repressing activity, but lost their activating function in vivo . Their DNA binding characteristics were consistent with an inability to respond to acetylserine by a qualitative change in the DNA-protein interaction . Three of the single residue substitutions resulted in constitutive activity of CysB . The electrophoretic mobility of the complex formed by one of the CysBc variants with the cysP promoter suggested a dimeric state of this protein . Characteristics of six truncated CysB variants lacking 5-30 C-terminal residues indicated the involvement of the C terminus in the DNA binding, oligomerization, and stability of CysB . The single substitution Y27G resulted in the CysBpc variant, able to bind DNA and to respond to the inducer by a qualitative change in the DNA-protein complex, but defective in the positive control of the cysP promoter. Drug Metab Dispos, 2000 Nov, 28(11), 1327 - 34 Automated definition of the enzymology of drug oxidation by the major human drug metabolizing cytochrome P450s; McGinnity DF et al.; A fully automated assay to determine the enzymology of drug oxidation by the major human hepatic cytochrome P450s (CYPs; CYP1A2, -2C9, -2C19, -2D6, and -3A4) coexpressed functionally in Escherichia coli with human NADPH-P450 reductase has been developed and validated . Ten prototypic substrates were chosen for which clearance was primarily CYP-dependent, and the activities of these five major CYPs were represented . A range of intrinsic clearance (CL(int)) values were obtained for substrates in both pooled human liver microsomes (HLM; 1-380 microl . min(-1)mg(-1)) and recombinant CYPs (0.03-7 microl . min(-1)pmol(-1)) and thus the percentage contribution of individual CYPs toward their oxidative metabolism could be estimated . All the assignments were consistent with the available literature data . Tolbutamide was metabolized by CYP2C9 (70%) and CYP2C19 (30%), diazepam by CYP2C19 (100%), ibuprofen by CYP2C9 (90%) and CYP2C19 (10%), and omeprazole by CYP2C19 (68%) and CYP3A4 (32%) . Metoprolol and dextromethorphan were primarily CYP2D6 substrates and propranolol was metabolized by CYP2D6 (59%), CYP1A2 (26%), and CYP2C19 (15%) . Diltiazem, testosterone, and verapamil were metabolized predominantly by CYP3A4 . In addition, the metabolite profile for the CYP-dependent clearance of several markers determined by mass spectroscopy was as predicted from the literature . There was a good correlation between the sum of individual CYP CL(int) and HLM CL(int) (r(2) = 0.8, P <.001) for the substrates indicating that recombinant CYPs may be used to predict HLM CL(int) data . This report demonstrates that recombinant human CYPs may be useful as an approach for the prediction of the enzymology of human CYP metabolism early in the drug discovery process. Chin J Biotechnol, 1999, 15(4), 225 - 30 Gene fusion and expression of heat-labile and heat-stable enterotoxins of enterotoxigenic Escherichia coli; Bing X et al.; The vaccine candidate comprising the genes that code the B subunit of the heat-labile enterotoxin (LT-B) and the heat-stable enterotoxin (ST) of enterotoxigenic E . coli (ETEC) had been constructed by recombinant genetic techniques . The 5'terminus of the gene encoding pro-ST was genetically fused to the 3'terminus of the LT-B gene . The pro-ST gene containing mature ST sequence and pro sequence which codes for the pro region of ST precursors was amplified by PCR from pSLN004 plasmid . To reduce toxicity of the ST in vitro was substituted Leu for Ala residue at position 14 of ST by oligonucleotide-directed site mutagenesis . For this construction, the expression of ST antigenicity and LT antigenicity were obtained when a five amino-acid or a nine-amino-acid linker were included between the LT-B and pro-ST moieties . The LT-B/pro-ST fusion peptides possessed no enterotoxic activity of heat-stable and heat-labile enterotoxins, and retained the ability to bind GM1 ganglioside . More importantly, these LT-B/pro-ST fusion peptides were immunogenic . The preparations containing the hybrid molecule elicited special antibodies that were to recognize native toxin in vitro. Res Microbiol, 2000 Sep, 151(7), 563 - 74 Effects of global regulatory proteins and environmental conditions on fimbrial gene expression of F165(1) and F165(2) produced by Escherichia coli causing septicaemia in pigs; Daigle F et al.; Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and possess at least two types of fimbriae . F165(1) fimbriae belong to the P fimbrial family and F165(2) fimbriae belong to the S fimbrial family . Regulatory regions of foo (F165(1)) and fot (F165(2)) fimbrial gene clusters from wild-type strain 4787 were sequenced and characterised . Expression of F165(1) and F165(2) fimbrial genes was analysed by using lacZ and/or luxAB as reporter genes under the control of the native fimbrial promoters . Differential expression of fimbrial genes was observed . Global regulatory mechanisms such as catabolite repression, leucine-responsive regulatory protein (Lrp), methylation and DNA supercoiling were demonstrated to influence foo and fot expression . foo and fot expression was optimal at 37 degrees C and under aerobic conditions . Expression of foo was higher on minimal medium, whereas fot expression was higher on complex Luria-Bertani medium . This could reflect an in vivo differential expression. Res Microbiol, 2000 Sep, 151(7), 535 - 46 Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene; Machado J et al.; A total of 182 strains of Escherichia coli (133 reference strains, 22 clinical strains, nine nonmotile strains and 18 strains derived from K-12) were characterized by HhaI restriction of the amplified flagellin gene (fliC) . The amplified fliC product was a single band between 0.9 and 2.6 kbp . With the collection of reference strains which represented 48 flagellar types (H-types), a total of 62 patterns (F-types) were observed after HhaI restriction . A single F-type was associated with each of 39 H-types and more than one F-type was associated with the other nine H-types . Antigenically related H-types 12 and 45 gave a single F-type . The determination of HhaI-fliC F-types could allow deduction of all H-types and subdivision of some of these . Application of this identification system to 22 E . coli clinical isolates yielded nine F-patterns and the deduced H-types were confirmed by serotyping in all cases . Nine nonmotile strains were studied and their F-types were also identified . The proposed determination of fliC restriction patterns should be helpful for epidemiological studies. J Biol Chem, 2001 Jan 5, 276(1), 386 - 94 DNA melting within a binary sigma(54)-promoter DNA complex; Cannon W et al.; The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription . Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes . Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site . We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I . It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity . Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex. Antimicrob Agents Chemother, 2000 Nov, 44(11), 3092 - 6 Expression and characterization of recombinant human-derived Pneumocystis carinii dihydrofolate reductase; Ma L et al.; Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia . While the rat-derived P . carinii DHFR has been well characterized, kinetic studies of human-derived P . carinii DHFR, which differs from rat-derived P . carinii DHFR by 38% in amino acid sequence, have not been reported to date . Here we report on the expression and kinetic characterization of the recombinant human-derived P . carinii DHFR . The 618-bp coding sequence of the human-derived P . carinii DHFR gene was expressed in Escherichia coli . As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence . Kinetic analysis showed that the K(m) values for dihydrofolate and NADPH were 2.7 +/- 0.3 and 14.0 +/- 4.3 microM, respectively, which are similar to those reported for rat-derived P . carinii DHFR . Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P . carinii DHFR, with K(i) values of 0.28 +/- 0.08 and 0.065 +/- 0.005 microM, respectively, while trimetrexate and methotrexate were potent inhibitors, with K(i) values of 0.23 +/- 0.03 and 0.016 +/- 0.004 nM, respectively . The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for human-derived P . carinii DHFR. Protein Expr Purif, 2000 Oct, 20(1), 128 - 31 One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase; Soo Choi W et al.; A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described . PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex . The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads . An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture . PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA . Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells . The preparation contained a small amount of a "nicked" form of PDPc . The cleavage is between Arg-394 and Arg-395 . Protein Expr Purif, 2000 Oct, 20(1), 124 - 7 Functional recombinant rabbit muscle phosphoglucomutase from Escherichia coli; Chae YK et al.; The gene coding for phosphoglucomutase (PGM) from Oryctolagus cuniculus (rabbit) has been expressed in Escherichia coli under a T7 expression system with a His-tag . About half of the expressed PGM protein was present in inclusion bodies, but this protein was inactive when solubilized . The protein in the soluble cell fraction was isolated and purified in one step on a Ni-NTA column . The eluate from this column was adjusted to 95% saturated ammonium sulfate, and the resulting protein precipitate was resuspended in sodium phosphate buffer and dialyzed against 2.5 M ammonium sulfate . The final yield of protein was about 10 mg per liter of LB medium . The protein was judged to be greater than 90% pure on the basis of gel electrophoresis and activity measurements (128 U per milligram) . Our motivation for developing this bacterial production system for PGM has been to prepare sufficient quantities of stable-isotope-labeled protein for experiments that utilize recently developed NMR technologies suitable for proteins the size of PGM (61.6 kDa) . Preliminary NMR studies indicate that the current level of purity is adequate for this work . The construct described here was designed to incorporate an N-terminal His-tag for ease of isolation . Although PGM is a metalloprotein, the His-tag does not appear to interfere with activity . The presence of the His-tag should not pose a problem for proposed (31)P NMR investigations of the protein and its complexes in aqueous solution or incorporated into reverse micelles . However, we plan to design a cleavable His-tag for later (1)H, (13)C, (15)N studies of the active site, which includes essential histidine residues . Protein Expr Purif, 2000 Oct, 20(1), 81 - 6 Recombinant maize protoporphyrinogen IX oxidase expressed in Escherichia coli forms complexes with GroEL and DnaK chaperones; de Marco A et al.; The clone corresponding to maize plastidic protoporphyrinogen IX oxidase (PPO) has been isolated by functional complementation and inserted into a pET16b vector for expression in Escherichia coli . Recombinant PPO was purified by standard affinity chromatography using a metal chelating resin . Two contaminants copurified with recombinant PPO and were identified as GroEL and DnaK . Since chaperone binding to hydrophobic regions of the protein is regulated by ATP availability, an ATP washing step was introduced prior to elution of the recombinant protein from an affinity column . This washing step selectively removed both chaperones and allowed the recovery of pure PPO . Coexpression of PPO and GroELS resulted in a sixfold increase of soluble PPO yield, suggesting that bacterial chaperones could be limiting during the folding of the heterologous protein . However, a portion of PPO was still found in the insoluble fraction . Buffer containing the GroEL and DnaK enabled resuspension of PPO from the insoluble fraction but failed to enhance refolding of the denaturated protein . Attempts to increase the amount of soluble PPO using a thioredoxin-PPO fusion protein were not successful . Initial characterization of the recombinant PPO found that it possessed a high V(max), an elevated affinity for substrate, and an elevated sensitivity to PPO inhibitor herbicides compared to previous reports . Protein Expr Purif, 2000 Oct, 20(1), 73 - 80 Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli; Shimada T et al.; Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced . Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E . coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems . In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5 . Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E . coli coexpressing CYP1B1 and the reductase proteins . Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E . coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required . However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5 . These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes . Protein Expr Purif, 2000 Oct, 20(1), 66 - 72 Purification of myristoylated and nonmyristoylated neuronal calcium sensor-1 using single-step hydrophobic interaction chromatography; Fisher JR et al.; Neuronal calcium sensors (NCSs) belong to a family of Ca(2+)-binding proteins, which serve important functions in neurotransmission, and are highly conserved from yeast to humans . Overexpression of the neuronal calcium sensor-1, called frequenin in the fruit fly and in frog, increases the release of neurotransmitters . Studying the functional role of frequenin in mammals and understanding its structural dynamics is critically dependent on the availability of active purified protein . Neuronal calcium sensors like other members of the family share common structural features: they contain four EF-hands as potential binding sites for Ca(2+) and an N-terminal consensus sequence for myristoylation . Previously, recoverin, distantly related to NCSs, has been expressed and purified from Escherichia coli, involving a combination of different chromatographic steps . NCS-1 has earlier been purified adopting a two-step procedure used for recoverin purification . We have overexpressed NCS-1 from rat in its myristoylated and nonmyristoylated form in E . coli and purified it from crude lysates using a single-step hydrophobic interaction chromatography . The purified protein was identified by Western blotting and mass spectrometry and assayed for its ability to bind Ca(2+) using a Ca(2+) shift assay, terbium fluorescence, and Stains-all binding . The present protocol provides a rapid, more efficient and simplified, single-step method for purifying NCS-1 for structural and functional studies . This method can also be applied to purify related proteins of the superfamily . Protein Expr Purif, 2000 Oct, 20(1), 21 - 6 The delta-endotoxin proteins accumulate in Escherichia coli as a protein-DNA complex that can be dissociated by hydrophobic interaction chromatography; Chaturvedi R et al.; The insecticidal protein CryIAc accumulated to form inclusion bodies in Escherichia coli upon overexpression of the cloned gene . The solubilized inclusion bodies contained the delta-endotoxin in association with DNA fragments of about 25 kb . The protein-DNA complex could be dissociated and the delta-endotoxin purified by hydrophobic interaction chromatography on phenyl-Sepharose . The DNA was washed out in the high-salt buffer while the delta-endotoxin was bound to the matrix and was eluted at 4 degrees C by a stepwise decreasing potassium chloride gradient . The DNA-protein complex also contained plasmids harbored by the host strain . The plasmid DNA associated with the complex became competent to transform E . coli only after it was dissociated from the delta-endotoxin . The hydrophobic interaction chromatography provides an efficient method for the purification of DNA-free activated toxin . Protein Expr Purif, 2000 Oct, 20(1), 10 - 20 Expression in Escherichia coli, folding in vitro, and characterization of the carbohydrate recognition domain of the natural killer cell receptor NKR-P1A; Kogelberg H et al.; NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NK-like T cells and is an activator of cytotoxicity . Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coli and folded in vitro . The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure . A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure . The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry . These are characteristic of a long form CRD . The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the (15)N-labeled CRD are those of a folded protein . Chemical shifts of H(alpha) and NH protons indicate a considerable amount of beta-strand structure . Successful folding in the absence of Ca(2+), coupled with the lack of chemical shift changes upon addition of Ca(2+), suggests that the NKR-P1A-CRD may not be a Ca(2+)-binding protein . Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 11966 - 71 Polynucleotide phosphorylase functions both as a 3' right-arrow 5' exonuclease and a poly(A) polymerase in Escherichia coli; Mohanty BK et al.; In vitro, polynucleotide phosphorylase of Escherichia coli can both synthesize RNA by using nucleotide diphosphates as precursors and exonucleolytically degrade RNA in the presence of inorganic phosphate . However, because of the high in vivo concentration of inorganic phosphate in exponentially growing cells, it has been assumed that the enzyme works exclusively as an exonuclease . Here we demonstrate that, contrary to this prediction, polynucleotide phosphorylase not only synthesizes long, highly heteropolymeric tails in vivo, but also accounts for all of the observed residual polyadenylylation in poly(A) polymerase I deficient strains . In addition, the enzyme is responsible for adding the C and U residues that are found in poly(A) tails in exponentially growing cultures of wild type E . coli. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12020 - 5 Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble; Pan H et al.; To explore how distal mutations affect binding sites and how binding sites in proteins communicate, an ensemble-based model of the native state was used to define the energetic connectivities between the different structural elements of Escherichia coli dihydrofolate reductase . Analysis of this model protein has allowed us to identify two important aspects of intramolecular communication . First, within a protein, pair-wise couplings exist that define the magnitude and extent to which mutational effects propagate from the point of origin . These pair-wise couplings can be identified from a quantity we define as the residue-specific connectivity . Second, in addition to the pair-wise energetic coupling between residues, there exists functional connectivity, which identifies energetic coupling between entire functional elements (i.e., binding sites) and the rest of the protein . Analysis of the energetic couplings provides access to the thermodynamic domain structure in dihydrofolate reductase as well as the susceptibility of the different regions of the protein to both small-scale (e.g., point mutations) and large-scale perturbations (e . g., binding ligand) . The results point toward a view of allosterism and signal transduction wherein perturbations do not necessarily propagate through structure via a series of conformational distortions that extend from one active site to another . Instead, the observed behavior is a manifestation of the distribution of states in the ensemble and how the distribution is affected by the perturbation. Infect Immun, 2000 Nov, 68(11), 6472 - 7 Contribution of plasmid-encoded fimbriae and intimin to capacity of rabbit-specific enteropathogenic Escherichia coli to attach to and colonize rabbit intestine; Krejany EO et al.; Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC) . Fimbriae and intimin, the outer membrane protein product of the chromosomal eae gene, contribute to this process, but their relative roles and the nature of their interaction are not known . The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits . To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H-), in which the ralE and eae genes were insertionally inactivated . These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy . The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC . Nevertheless, a delta eae Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its eae-positive parent and far more extensively than an eae(+) delta ral strain . To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined . The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a delta eae delta ralE double mutant was incapable of colonizing the intestine . Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor . When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin. Infect Immun, 2000 Nov, 68(11), 6423 - 30 Involvement of focal adhesion kinase in Escherichia coli invasion of human brain microvascular endothelial cells; Reddy MA et al.; Escherichia coli K1 traversal across the blood-brain barrier is an essential step in the pathogenesis of neonatal meningitis . We have previously shown that invasive E . coli promotes the actin rearrangement of brain microvascular endothelial cells (BMEC), which constitute a lining of the blood-brain barrier, for invasion . However, signal transduction mechanisms involved in E . coli invasion are not defined . In this report we show that tyrosine kinases play a major role in E . coli invasion of human BMEC (HBMEC) . E . coli induced tyrosine phosphorylation of HBMEC cytoskeletal proteins, focal adhesion kinase (FAK), and paxillin, with a concomitant increase in the association of paxillin with FAK . Overexpression of a dominant interfering form of the FAK C-terminal domain, FRNK (FAK-related nonkinase), significantly inhibited E . coli invasion of HBMEC . Furthermore, we found that FAK kinase activity and the autophosphorylation site (Tyr397) are important in E . coli invasion of HBMEC, whereas the Grb2 binding site (Tyr925) is not required . Immunocytochemical studies demonstrated that FAK is recruited to focal plaques at the site of bacterial entry . Consistent with the invasion results, overexpression of FRNK, a kinase-negative mutant (Arg454 FAK), and a Src binding mutant (Phe397 FAK) inhibited the accumulation of FAK at the bacterial entry site . The overexpression of FAK mutants in HBMEC also blocked the E . coli-induced tyrosine phosphorylation of FAK and its association with paxillin . These observations provide evidence that FAK tyrosine phosphorylation and its recruitment to the cytoskeleton play a key role in E . coli invasion of HBMEC. Infect Immun, 2000 Nov, 68(11), 6115 - 26 The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli; Elliott SJ et al.; Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli (EHEC) is incompletely understood . In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters . We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin . Ler is therefore central to the process of attaching and effacing (AE) lesion formation . Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties . In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC . delta ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae . Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC. Free Radic Biol Med, 2000 Aug, 29(3-4), 357 - 67 A simpler, more robust method for the analysis of 8-oxoguanine in DNA; Beckman KB et al.; The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine . There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation . Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases . In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC . Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput . Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods. J Biotechnol, 2001 Nov 17, 84(1), 53 - 62 Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli; Greiner R et al.; Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established . High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E . coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P . Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E . coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E . coli are not identical . The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E . coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973 . The phytases: II . Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2) . Biochim . Biophys . Acta 302, 326-328) . The data demonstrate that the phytate-degrading enzyme P2 of E . coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5). J Biotechnol, 2001 Nov 17, 84(1), 27 - 32 Expression-independent consumption of substrates in cell-free expression system from Escherichia coli; Kim RG et al.; In a cell-free expression system derived from Escherichia coli, expression is abruptly ceased after 30 min of incubation while at this time not all the substrates have been utilized in expression . Expression-independent consumption of phosphoenolpyruvate and cysteine was found in this system, which was responsible for the above sudden cessation of expression . The above consumption was at least partially due to the dephosphorylation of nucleoside triphosphates and the conversion of cysteine into gamma-glutamylcysteine, respectively . Based on these, we developed a new system employing new S30 extract of lower phosphatase activity, higher cysteine concentration, and an inhibitor of glutathione synthesis pathway . This system showed 70% enhance in productivity (179-302 microg chloramphenicolacetyltransferase protein per ml reaction mixture per hour) over the model system. Mutat Res, 2000 Nov 6, 454(1-2), 71 - 6 Mutagenic effect by phenylalanine during gamma-irradiation of plasmid DNA in aqueous solution under oxic conditions; Reitsma-Wijker CA et al.; Irradiation of DNA in aqueous solution or in cells with gamma-rays results in different mutational spectra, indicating that in both situations different patterns of DNA damages are induced . One of the causes for these different types of damages might be the formation of secondary, organic radicals, if cells are irradiated . Some organic compounds, including the amino acid phenylalanine, are well known to produce radicals during irradiation . Under oxic conditions these secondary radicals react with oxygen, thus forming peroxyl radicals which can be very harmful to DNA, and which may, therefore, induce DNA damage leading to mutations . This study examines the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions . The results indicate that the formation of phenylalanine radicals influences the types of induced mutations in the gamma-radiation-induced mutation spectrum . The most prominent difference is the increase in G:C to T:A transversions and the decrease in G:C to A:T transitions in the presence of phenylalanine . Further, it appears that the gamma-radiation-induced mutation spectrum after irradiation of DNA in aqueous solution is more comparable to the intracellular gamma-radiation-induced mutation spectrum in E . coli cells, if phenylalanine is present during irradiation . Therefore, these results suggest that the presence of phenylalanine during irradiation of DNA in aqueous solution gives a better impression of gamma-radiation-induced mutations in bacterial systems than water only. Mutat Res, 2000 Nov 6, 454(1-2), 1 - 10 Differential mutation of transgenic and endogenous loci in vivo; Cosentino L et al.; Although chemicals usually induce very similar frequencies of mutations in transgenes and endogenous genes in vivo when given acutely, chronic exposure to N-ethyl-N-nitrosourea (ENU) produced a more complex pattern in which the endogenous locus was spared many mutations . Here, we demonstrate that the effect is neither ENU-specific nor locus-specific, and thus, may be important in the extrapolations of risk assessment and in understanding mutational mechanisms . During chronic mutagen exposure, mutations at the transgene accumulate linearly with time, i.e . in direct proportion to the dose received . In contrast, mutations at the endogenous gene are much less frequent than those of the transgene early in the exposure period and the accumulation is not linear with time, but rather accelerates as the exposure continues . Previous comparisons involved the endogenous Dlb-1 locus and the lacI transgene from the Big BlueMouse in the small intestine . These experiments involved the Dlb-1 locus and the lacZ transgene from the MutaMouse in the small intestine and the hprt locus and the lacZ transgene in splenocytes . Comparisons were made in both tissues after acute and chronic exposures to ENU, the original mutagen, and in the small intestine after exposures to benzo(a)pyrene . All comparisons showed that during chronic exposures mutations at the transgene accumulate linearly with the increasing duration of exposure, whereas induced mutations of the endogenous gene initially accumulate at a slower rate . Thus, the difference in mutational response observed during low chronic treatment is not unique to a particular transgene, endogenous gene, tissue, or mutagen used, but may be a general phenomenon of such genes. J Immunol, 2000 Oct 15, 165(8), 4379 - 87 Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2; Woodfolk JA et al.; The 29-kDa subtilase homologue, Tri r 2, derived from the dermatophyte fungus Trichophyton rubrum, exhibits unique immunologic characteristics in its ability to elicit immediate (IH) and delayed-type (DTH) hypersensitivity skin tests in different individuals . Thus, Tri r 2 provides a model for comparing the T cell repertoire in subjects with distinct immune responses to a single Ag . Recombinant Tri r 2 produced as a GST fusion protein in Escherichia coli stimulated strong in vitro lymphoproliferative responses in 10 IH and 10 DTH responders . Patterns of T cell epitope recognition were compared between skin test groups using 28 overlapping peptides (each in 12 replicate wells) derived from Tri r 2 to stimulate T lymphocyte proliferation in vitro . Peptide 5 (P5; aa 41-60) induced the strongest response in DTH subjects and showed the largest difference between DTH and IH responders in proliferation (mean standardized index, 2.22 and 0.82, respectively; p = 0.0047) and number of positive wells (81 vs 12) . Responses to P5 were associated with diverse HLA haplotypes . These results showed that P5 contains an immunodominant epitope specifically associated with DTH and that this peptide is recognized in a permissive manner . Cross-validated linear discriminant analysis using T cell proliferative responses to two regions of Tri r 2 (aa 51-90 and 231-270) gave a 95% predictive accuracy for classification of subjects into IH or DTH groups . We conclude that different immune responses to Trichophyton are mediated by distinct T cell repertoires between individuals with IH and DTH reactions to Tri r 2. J Biol Chem, 2001 Jan 12, 276(2), 1335 - 44 Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by activating NF-kappa B via Ref-1; Daily D et al.; The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites . This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis . E . coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-kappaB activation . Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity . Although DA significantly reduced NF-kappaB binding activity, Grx2 could stimulate the binding of NF-kappaB to DNA by: (i) translocating NF-kappaB from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-kappaBalpha, and (ii) activating the binding of pre existing nuclear NF-kappaB . The DNA binding activity of NF-kappaB itself was essential for neuronal survival . Overexpression of I-kappaB dominant negative gene (I-kappaB-DeltaN) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2 . Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-kappaB binding activity . These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-kappaB. J Biol Chem, 2000 Dec 15, 275(50), 39631 - 9 Subunit structure of a mammalian ER/Golgi SNARE complex; Xu D et al.; SNAP receptor (SNARE) complexes bridge opposing membranes to promote membrane fusion within the secretory and endosomal pathways . Because only the exocytic SNARE complexes have been characterized in detail, the structural features shared by SNARE complexes from different fusion steps are not known . We now describe the subunit structure, assembly, and regulation of a quaternary SNARE complex, which appears to mediate an early step in endoplasmic reticulum (ER) to Golgi transport . Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity binding site for sec22b, an R-SNARE . The syntaxin 5 amino-terminal domain potently inhibits SNARE complex assembly . The ER/Golgi quaternary complex is remarkably similar to the synaptic complex, suggesting that a common pattern is followed at all transport steps, where three Q-helices assemble to form a high affinity binding site for a fourth R-helix on an opposing membrane . Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others assemble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1 . Finally, we demonstrate that the quaternary complex containing sec22b is not an in vitro entity only, but is a bona fide species in living cells. J Immunol, 2000 Oct 1, 165(7), 3849 - 59 A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain; Flicker S et al.; Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens . We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens . The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species . By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A . The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity . Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity . It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments . Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation . We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies. FEBS Lett, 2000 Sep 1, 480(2-3), 127 - 31 31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE; Glaubitz C et al.; The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation-magic-angle spinning nuclear magnetic resonance spectroscopy (CP-MAS NMR) . EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers . CP-MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane . A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein . This is also supported by the observed restricted motion of the bound ligand . The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions. FEMS Microbiol Lett, 2000 Sep 15, 190(2), 223 - 9 Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans; Niehaus F et al.; The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli . The 2181-bp open reading frame encodes a protein of 727 amino acids . A hypothetical membrane linker region was found to be cleaved during processing in E . coli . The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography . Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5 . More than 35% of enzymatic activity was detected even at 120 degrees C . The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C . Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose . The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products. FEBS Lett, 2000 Oct 13, 483(1), 47 - 51 NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation; Heyes DJ et al.; NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway . POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus . This enzyme (His(6)-POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented . Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide . Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis. FEBS Lett, 2000 Oct 13, 483(1), 21 - 6 Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper-coordinated water molecule . is the water molecule really necessary for the enzymatic mechanism? Sette M, Bozzi M, Battistoni A, Fasano M, Paci M, Rotilio G. The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy . The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases . Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule . In spite of these differences the enzymatic activity is still high . Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes . Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction . A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies. Chem Biol, 2000 Oct, 7(10), 765 - 72 Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases; Ehmann DE et al.; BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products . These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation . The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages . Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds . RESULTS: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains . Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC . Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity . CONCLUSIONS: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation. J Immunol Methods, 2000 Oct 20, 244(1-2), 117 - 31 Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies; van der Geld YM et al.; Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG) . Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG . Purification of PR3 is laborious and requires large amounts of granulocytes . Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA . The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies . Recombinant PR3 produced in E . coli (rcPR3), P . pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used . Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3 . In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products . By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera . By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products . Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively . All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA . The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA . The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA . All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by all anti-PR3 mAbs . In conclusion, rPR3 expressed in insect cells, HMC-1 and 293 cells is recognized by anti-PR3 antibodies, whereas conformational epitopes recognized by anti-PR3 mAbs and PR3-ANCA are not well preserved on rPR3 expressed in E . coli or P . pastoris. J Biol Chem, 2001 Jan 12, 276(2), 1538 - 44 Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase; Mitchell LW et al.; Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) . The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252 . In this study, E . coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized . Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices . (13)C NMR studies of {3,5-(13)C}porphobilinogen bound at the active sites of the E . coli PBGS and the mutants show only minor chemical shift differences, i.e . environmental alterations . Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E . coli PBGS shows eight spatially distinct and structurally equivalent subunits . Biochemical data for E . coli PBGS have been interpreted to support both four and eight active sites . A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry . Product binding studies with wild-type E . coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate . Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity. EMBO J, 2000 Oct 16, 19(20), 5542 - 51 Repair of chromosomal abasic sites in vivo involves at least three different repair pathways; Otterlei M et al.; We introduced multiple abasic sites (AP sites) in the chromosome of repair-deficient mutants of Escherichia coli, in vivo, by expressing engineered variants of uracil-DNA glycosylase that remove either thymine or cytosine . After introduction of AP sites, deficiencies in base excision repair (BER) or recombination were associated with strongly enhanced cytotoxicity and elevated mutation frequencies, selected as base substitutions giving rifampicin resistance . In these strains, increased fractions of transversions and untargeted mutations were observed . In a recA mutant, deficient in both recombination and translesion DNA synthesis (TLS), multiple AP sites resulted in rapid cell death . Preferential incorporation of dAMP opposite a chromosomal AP site ('A rule') required UmuC . Furthermore, we observed an 'A rule-like' pattern of spontaneous mutations that was also UmuC dependent . The mutation patterns indicate that UmuC is involved in untargeted mutations as well . In a UmuC-deficient background, a preference for dGMP was observed . Spontaneous mutation spectra were generally strongly dependent upon the repair background . In conclusion, BER, recombination and TLS all contribute to the handling of chromosomal AP sites in E.coli in vivo. EMBO J, 2000 Oct 16, 19(20), 5473 - 82 The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3; Peyroche G et al.; RNA polymerase I (Pol I) is dedicated to transcription of the large ribosomal DNA (rDNA) . The mechanism of Pol I recruitment onto rDNA promoters is poorly understood . Here we present evidence that subunit A43 of Pol I interacts with transcription factor Rrn3: conditional mutations in A43 were found to disrupt the transcriptionally competent Pol I-Rrn3 complex, the two proteins formed a stable complex when co-expressed in Escherichia coli, overexpression of Rrn3 suppressed the mutant phenotype, and A43 and Rrn3 mutants showed synthetic lethality . Consistently, immunoelectron microscopy data showed that A43 and Rrn3 co-localize within the Pol I-Rrn3 complex . Rrn3 has several protein partners: a two-hybrid screen identified the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by affinity chromatography . Our results suggest that Rrn3 plays a central role in Pol I recruitment to rDNA promoters by bridging the enzyme to the core factor . The existence of mammalian orthologues of A43 and Rrn3 suggests evolutionary conservation of the molecular mechanisms underlying rDNA transcription in eukaryotes. EMBO J, 2000 Oct 16, 19(20), 5353 - 61 Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli; Lee SJ et al.; The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator . No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG) . Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc) . We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside . Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region . Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars . The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation. EMBO J, 2000 Oct 16, 19(20), 5344 - 52 A novel regulatory role of glucose transporter of Escherichia coli: membrane sequestration of a global repressor Mlc; Tanaka Y et al.; External glucose stimulates transcription of several genes including ptsG encoding IICB(Glc), a membrane component of the phosphotransferase system (PTS), by relieving the negative regulation of a global repressor Mlc in Escherichia coli . We investigate here how glucose modulates Mlc action . The Mlc-mediated repression is eliminated by a ptsI mutation, while Mlc is constitutively active in a ptsG mutant . We show that IICB(Glc)-FLAG interacts physically with Mlc in crude extracts prepared from cells in which IICB(Glc) is supposed to exist as the non-phosphorylated form . The IICB(Glc)-Mlc interaction is no longer observed when IICB(Glc) is phosphorylated . Exogenously added purified Mlc binds to purified IICB(Glc)-FLAG . We also demonstrate that Mlc is associated with membrane when IICB(Glc) is dephosphorylated while it is in the cytoplasm when IICB(Glc) is phosphorylated or absent . We conclude that IICB(Glc) regulates the cellular localization of Mlc, depending on its phosphorylation state, which is determined by the availability of external glucose . Thus, glucose induces the transcription of Mlc-regulated promoters by sequestering Mlc to the membrane through dephosphorylation of IICB(Glc). Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 499 - 506 Cytotoxic activity of recombinant bFGF-rViscumin fusion proteins; Schmidt A et al.; A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized . bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM . rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM . Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure . The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM . Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB . We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development . Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 476 - 86 Differential gene expression in apoptosis: identification of ribosomal protein S29 as an apoptotic inducer; Khanna N et al.; To identify genes that are specifically involved in apoptosis, poly(A)(+) RNAs were isolated from untreated control rat thymocytes and from adriamycin-induced apoptotic thymocytes . Directionally cloned cDNA libraries were then constructed in UNIZAP-XR vectors followed by biotin-based subtractive hybridization . Three clones were confirmed to be differentially expressed by dot blotting . Sequence analysis revealed homology to two genes previously identified, whereas one clone was novel and did not have homology to any known sequence . One clone was identical to the ribosomal protein S29, and the other was homologous to L8 ribosomal protein . Northern blot analysis revealed a marked increase in the expression of mRNA encoding ribosomal protein S29 in the apoptotic thymocytes compared to the controls . Transfection studies revealed that enhanced S29 expression resulted in increased apoptosis in rat thymocytes and HeLa cells as assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, TUNEL, FACS, and internucleosomal DNA fragmentation . This was accompanied by upregulation of p53, Caspase 3, and bax, whereas bcl-2 was downregulated as revealed by Western blotting . The current findings provide the first hint of a role for ribosomal protein S29 in the apoptotic process . Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 443 - 7 Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions; Das KC et al.; Thioredoxin is a ubiquitous small protein known to protect cells and tissues against oxidative stress . However, its exact antioxidant nature has not been elucidated . In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger . Human thioredoxin at 3 microM caused 50% inhibition of TEMP-(1)O(2) (TEMPO) adduct formation in a photolysis EPR study . In contrast, Escherichia coli thioredoxin caused 50% inhibition of TEMPO formation at 80 microM . Both E . coli thioredoxin and human thioredoxin inhibited (*)OH dependent DMPO-OH formation as demonstrated by EPR spectrometry . The quenching of (1)O(2) or scavenging of (*)OH was not dependent upon the redox state of thioredoxin . Using a human thioredoxin in which the structural cysteines were mutated to alanine, Trx-C3A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scavenging properties . In addition, using a quadruple mutant Trx-C4A, where one of the catalytic cysteines, C35 was mutated to alanine in addition to the mutated structural cysteines, we demonstrated that catalytic cysteines are also required for the scavenging action of thioredoxin . Identification of thioredoxin as a (1)O(2) quencher and (*)OH scavenger may be of significant importance in explaining various redox-related antioxidant functions of thioredoxin . J Biol Chem, 2001 Jan 19, 276(3), 2276 - 85 Epub 2000 Oct 16. Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts; Sung JS et al.; The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay . Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue . The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E . coli cells . In reactions utilizing E . coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells . The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity . Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides . Reactions conducted with E . coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair . The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug . The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum . In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity. Arch Biochem Biophys, 2000 Sep 15, 381(2), 317 - 22 Redox regulation of yeast flavin-containing monooxygenase; Suh JK et al.; The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione . The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment . We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential . The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER . Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation . Cys 353 is the principal residue in the redox-sensitive switch . We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO . The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory. Arch Biochem Biophys, 2000 Sep 15, 381(2), 313 - 6 Novel nonpeptidic inhibitors of peptide deformylase; Jayasekera MM et al.; A novel series of nonpeptidic compounds structurally related to the known anticholesteremic thyropropic acid were found to inhibit Escherichia coli peptide deformylase (PDF), with IC50 values in the low-micromolar range . Kinetic analysis of {4-(4-hydroxyphenoxy)-3,5-diiodophenyl}acetic acid reveals competitive inhibition, with a Ki value of 0.66 +/- 0.007 microM . A structure-activity relationship study demonstrates that the carboxylate is required for activity, while the distal phenolic function can be methylated without significant effect . Either decreasing the number of iodine atoms on the molecule to one or increasing the number of iodine atoms to four results in the loss of an order of magnitude in potency . These compounds are the first nonpeptidic inhibitors disclosed and represent a template from which better inhibitors might be designed. Arch Biochem Biophys, 2000 Sep 15, 381(2), 173 - 80 Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L; Mercke P et al.; In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate . The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases) . In Artemisia annua L . (annual wormwood), a number of such sesquiterpene cyclases are active . We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis . This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence . The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms . When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate . GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively) . Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes . The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5 . A putative reaction mechanism for amorpha-4,11-diene synthase is suggested. J Interferon Cytokine Res, 2000 Sep, 20(9), 779 - 85 Canine interleukin-13: molecular cloning of full-length cDNA and expression of biologically active recombinant protein; Yang S et al.; Interleukin-13 (IL-13) regulates immune responses mediated by type 2 T helper lymphocytes (Th2) in the human and mouse . To study the function of this cytokine in the dog, we have isolated a cDNA that encodes the full-length canine IL-13 (CaIL-13) precursor polypeptide of 131 amino acids . CaIL-13 shares significant homology with the IL-13 amino acid sequences of cattle (54.1%), mouse (39.6%), and rat (36.6%) but shares the highest identity with human IL-13 (HuIL-13) (61.8%) . The predicted CaIL-13 mature polypeptide of 111 residues was expressed in bacteria, and recombinant CaIL-13 (rCaIL-13) was isolated from inclusion bodies and refolded . rCaIL-13 stimulated the proliferation of TF-1 cells, which are derived from human erythroleukemia cells and respond to IL-13 as well as to a number of other human and murine cytokines . CaIL-13 mRNA was readily detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells from lymph nodes and peripheral blood . The gene sequence and biologically active recombinant protein for CaIL-13 will be useful reagents to determine the role of IL-13 in the regulation of canine immune responses. Radiats Biol Radioecol, 2000 Jul-Aug, 40(4), 416 - 9 {The evaluation of the role of recovery from potentially lethal and sublethal damages in the RBE of densely ionizing radiation}; Shvedenko VI et al.; The new data confirming the relations between RBE and recovery of cells are presented . The quantitative evaluation of the contribution of potentially lethal and sublethal damage recovery in radiosensitivity of cells of various origin after exposure to low- and high-LET ionizing radiation was carried out . The conclusion about the greater contribution of potentially lethal damage recovery in the magnitude of RBE in comparison with sublethal damage recovery was made. J Biol Chem, 2000 Dec 22, 275(51), 39823 - 6 Cloning and recombinant expression of a structurally novel human secreted phospholipase A2; Gelb MH et al.; Mammals contain a diverse set of secreted phospholipases A(2) (sPLA(2)s) that liberate arachidonic acid from phospholipids for the production of eicosanoids and exert a variety of physiological and pathological effects . We report the cloning, recombinant expression, and kinetic properties of a novel human sPLA(2) that defines a new structural class of sPLA(2)s called group XII . The human group XII (hGXII) cDNA contains a putative signal peptide of 22 residues followed by a mature protein of 167 amino acids that displays homology to all known sPLA(2)s only over a short stretch of amino acids in the active site region . Northern blot and reverse transcription-polymerase chain reaction analyses show that the tissue distribution of hGXII is distinct from the other human sPLA(2)s with strong expression in heart, skeletal muscle, kidney, and pancreas and weaker expression in brain, liver, small intestine, lung, placenta, ovaries, testis, and prostate . Catalytically active hGXII was produced in Escherichia coli and shown to be Ca(2+)-dependent despite the fact that it is predicted to have an unusual Ca(2+)-binding loop . Similar to the previously characterized mouse group IIE sPLA(2)s, the specific activity of hGXII is low in comparison to that of other mammalian sPLA(2), suggesting that hGXII could have novel functions that are independent of its phospholipase A(2) activity. J Mol Biol, 2000 Oct 27, 303(3), 423 - 32 NMR structure of oxidized glutaredoxin 3 from Escherichia coli; Nordstrand K et al.; A high precision NMR structure of oxidized glutaredoxin 3 {C65Y} from Escherichia coli has been determined . The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage . A comparison with the previously determined structure of glutaredoxin 3 {C14S, C65Y} in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue . In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione . The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide . This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides . J Mol Biol, 2000 Oct 27, 303(3), 383 - 93 pH-controlled quaternary states of hexameric DnaB helicase; Donate LE et al.; DnaB is the major helicase in the Escherichia coli replisome . It is a homohexameric enzyme that interacts with many other replisomal proteins and cofactors . It is usually loaded onto a single strand of DNA at origins of replication from its complex with its loading partner DnaC, then translocates in the 5' to 3' direction, unwinding duplex DNA in an NTP-driven process . Quaternary polymorphism has been described for the DnaB oligomer, a feature it has in common with some other hexameric helicases . In the present work, electron microscopy and in- depth rotational analysis studies of negatively stained specimens has allowed the establishment of conditions that govern the transition between the two different rotational symmetry states (C(3) and C(6)) of DnaB . It is shown: (a) that the pH value of the sample buffer, within the physiological range, dictates the quaternary organisation of the DnaB oligomer; (b) that the pH-induced transition is fully reversible; (c) that the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB.DnaC complex exists only as particles with C(3) symmetry; and (e) that DnaC interacts only with DnaB particles that have C(3) symmetry . Structural consequences of this quaternary polymorphism, as well as its functional implications for helicase activity, are discussed . J Mol Biol, 2000 Oct 27, 303(3), 371 - 82 Two novel flagellar components and H-NS are involved in the motor function of Escherichia coli; Ko M et al.; A mutation in H-NS results in non-flagellation of Escherichia coli due to a reduced expression of the flhDC master operon . We found that the hns-negative strain restored its flagellation in the presence of flhDC, although the resulting strain was still non-motile . Since the intracelluar levels of motor components MotA, MotB, and FliG in the Deltahns strain were unaltered, the non-motility indicates that H-NS affects flagellar function as well as biogenesis . We obtained an insertion in ycgR, a putative gene encoding a protein of 244 amino acid residues, which suppresses the motility defect of hns-deficient cells . The abnormally low swimming speed of hns mutant cells was fully restored by an insertion in ycgR, as assessed with computer-assisted motion analysis . A similar suppressor phenotype was observed with a multicopy expression of yhjH, a putative gene encoding a polypeptide of 256 amino acid residues . Since the flagella of most hns-deficient cells were not rotating, except a few with reduced speed, the suppression appears to increase the number of rotating flagella as observed with tethered bacteria . The ycgR and yhjH genes contain the consensus sequence found among the class III promoters of the flagellar regulon, and their expression monitored with a lacZ fusion requires FlhDC . These findings suggest that ycgR and yhjH, together with H-NS, are involved in the motor function and constitute new members of the flagellar regulon . J Org Chem, 2000 Oct 20, 65(21), 6904 - 9 Synthesis of gallic acid: Cu(2+)-mediated oxidation of 3-dehydroshikimic acid; Kambourakis S et al.; With the elaboration of high-yielding, high-titer syntheses of 3-dehydroshikimic acid from glucose using recombinant Escherichia coli, oxidation of this hydroaromatic becomes a potential route for synthesis of gallic acid . Conversion of 3-dehydroshikimic acid into gallic acid likely proceeds via initial enolization of an alpha-hydroxycarbonyl and oxidation of the resulting enediol . 3-Dehydroshikimate enolization in water was catalyzed by inorganic phosphate while Zn(2+) was used to catalyze enolization in acetic acid . Enediol oxidation employed Cu(2+) as either the stoichiometric oxidant or as a catalyst in the presence of a cooxidant . Gallic acid was produced in a yield of 36% when 3-dehydroshikimic acid in phosphate-buffered water reacted for 35 h with H2O2 and catalytic amounts of CuSO(4) . 3-Dehydroshikimate-containing, phosphate-buffered culture supernatants reacted with stoichiometric amounts of CuCO(3)Cu(OH)(2) and Cu(x)(H(3-x)(PO4)(2) to give gallic acid in yields of 51% in 5 h and 43% in 12 h, respectively . Solutions of 3-dehydroshikimic acid in acetic acid reacted with stoichiometric amounts of Cu(OAc)(2) to afford a 74% yield of gallic acid in 36 h . Acetic acid solutions of 3-dehydroshikimic acid could also be oxidized by air using catalytic quantities of Cu(OAc)(2) . ZnO accelerated these oxidations leading to a 67% yield of gallic acid in 4 h when an acetic acid solution of 3-dehydroshikimic acid was reacted with O(2) and a catalytic amount of Cu(OAc)(2). Biol Chem, 2000 Aug, 381(8), 695 - 703 Plant methionine synthase: new insights into properties and expression; Eckermann C et al.; We investigated the enzyme methionine synthase (MSY) in Catharanthus roseus . The properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in Escherichia coli . The protein was a monomer and accepted both the triglutamate (CH3-H4PteGlu3, apparent Km = 80 microM) and the monoglutamate (CH3-H4PteGlu1, apparent Km = 350 microM) of methyl-5,6,7,8-tetrahydropteroate as methyl donor, with a ratio of approximately 90:1 in favor of the triglutamate . Both activities required inorganic phosphate, but with different kinetics, and both were dependent on reducing agents . The activity required zinc, as shown by depletion and reconstitution experiments . Mg2+ had no effect on the activity . Two MSY isoforms purified from parsley cell cultures revealed the same properties as the C . roseus enzyme, however, the parsley proteins had no detectable activity with the monoglutamate substrate . The second part of the work compared the expression of the three enzymes of the methyl cycle (MSY, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase) . In cell cultures, all three enzymes were present under all conditions investigated, with small changes at the protein level and more pronounced changes at the RNA level . Studies with seedlings revealed a low expression of all three enzymes in cotyledons, when compared to hypocotyls and radiculas . Immunohistochemical experiments indicated that MSY expression in cotyledons is cell-type specific, with the strongest signals detected in the upper epidermis. Biol Chem, 2000 Aug, 381(8), 667 - 78 Two differentially regulated class II chitinases from parsley; Ponath Y et al.; Two distinct cDNA clones, PcCHI1 and PcCHI2, with high sequence similarity to plant chitinases were isolated from parsley (Petroselinum crispum), expressed in Escherichia coli, and the encoded proteins functionally identified as endochitinases . Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development . Infection of parsley leaf buds with Phytophthora sojae resulted in the rapid, transient and highly localized accumulation of PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 mRNA and protein were systemically induced at later infection stages . Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells . In uninfected plants, PcCHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth . Localization of PcCHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue . In addition to such distinct roles of PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development. Mol Cell, 2000 Sep, 6(3), 661 - 71 Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion; Sato TK et al.; In yeast, the Class C Vps protein complex (C-Vps complex), composed of Vps11, Vps16, Vps18, and Vps33, functions in Golgi-to-vacuole protein transport . In this study, we characterized and purified this complex and identified its interaction with the syntaxin homolog Vam3 . Vam3 pairs with the SNAP-25 homolog Vam7 and VAMP homolog Vti1 to form SNARE complexes during vesicle docking/fusion with the vacuole . The C-Vps complex does not bind to Vam3-Vti1-Vam7 paired SNARE complexes but instead binds to unpaired Vam3 . Antibodies to a component of this complex inhibited in vitro vacuole-to-vacuole fusion . Furthermore, temperature-conditional mutations in the Class C VPS genes destabilized Vam3-Vti1-Vam7 pairing . Therefore, we propose that the C-Vps complex associates with unpaired (activated) Vam3 to mediate the assembly of trans-SNARE complexes during both vesicle docking/fusion and vacuole-to-vacuole fusion. Mol Cell, 2000 Sep, 6(3), 583 - 92 Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament; Mazin AV et al.; Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes . Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially . Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament . Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament . Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54 . We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process. Mol Cell, 2000 Sep, 6(3), 573 - 82 Tn7 transposes proximal to DNA double-strand breaks and into regions where chromosomal DNA replication terminates; Peters JE et al.; We report that the bacterial transposon Tn7 can preferentially transpose into regions where chromosomal DNA replication terminates . DNA double-strand breaks are associated with the termination of chromosomal replication; therefore, we directly tested the effect of DNA breaks on Tn7 transposition . When DNA double-strand breaks are induced at specific sites in the chromosome, Tn7 transposition is stimulated and insertions are directed proximal to the induced break . The targeting preference for the terminus of replication and DNA double-strand breaks is dependent on the Tn7-encoded protein TnsE . The results presented in this study could also explain the previous observation that Tn7 is attracted to events associated with conjugal DNA replication during plasmid DNA transfer. J Enzyme Inhib, 2000, 15(5), 471 - 86 Carbonic anhydrase activators: synthesis of high affinity isozymes I, II and IV activators, derivatives of 4-(arylsulfonylureido-amino acyl)ethyl-1H-imidazole; Supuran CT et al.; Based on the X-ray crystallographic structure of the adduct of human carbonic anhydrase II (hCA II) with the weak activator histamine (Briganti, F., Mangani, S., Orioli, P., Scozzafava, A., Vernaglione, G . and Supuran, C.T . (1997) Biochemistry, 36, 10,384-10,392), a novel class of tight-binding CA activators was designed by using histamine (Hst) as lead molecule . Thus, N-1-tritylsulfenyl Hst was synthesized by reaction of Hst with tetrabromophthalic anhydride followed by protection of its imidazole moiety with tritylsulfenyl chloride . After hydrazinolysis, it afforded a key intermediate which was derivatized at the aliphatic amino group . Reaction of the key intermediate with 4-fluorophenylsulfonylureido amino acids (fpu-AA) or 2-toluenesulfonylureido amino acids (ots-AA) in the presence of carbodiimides, afforded after deprotection, a series of compounds with the general formula fpu/ots-AA-Hst (fpu = 4-FC6H4SO2NHCO; ots = 2-MeC6H4SO2NHCO) . Some structurally related dipeptides with the general formula fpu/ots-AA1-AA2-Hst (AA, AA1 and AA2 represent amino acyl moieties), were also prepared, by a strategy similar to that used for the simple amino acyl compounds above . The new derivatives proved to be efficient in vitro activators of three CA isozymes . Best activity was shown against hCA I and bCA IV, for which some of the new compounds (such as the Lys, Arg, His or the dipeptide derivatives) showed affinities in the 2-12 nm range (h = human; b = bovine isozymes) . hCA II was on the other hand somehow less prone to activation by the new derivatives, which possessed affinities around 30-60 nM for this isozyme . Ex vivo experiments showed some of the new activators to strongly enhance red cell CA activity (180-230%) after incubation with human erythrocytes . This new class of CA activators might lead to the development of drugs/diagnostic tools for the CA deficiency syndrome, a genetic disease of bone, brain and kidneys. Mol Microbiol, 2000 Oct, 38(1), 167 - 75 The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA; Schneider R et al.; The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA . Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling . Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling . Any deviations from these optimal levels decrease fis promoter activity . This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E . coli. Mol Microbiol, 2000 Oct, 38(1), 126 - 39 A role for the Escherichia coli H-NS-like protein StpA in OmpF porin expression through modulation of micF RNA stability; Deighan P et al.; When a wild-type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two-dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary . One of these was identified as the outer membrane porin protein, OmpF . In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly . Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96% . This post-transcriptional expression was found to result from a strong reduction in the half-life of ompF message in the double mutant . The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF . Moreover, micF antisense RNA accumulated considerably in an stpA hns background . Transcriptional data from a micF-lacZ fusion and measurements of micF RNA half-life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone) . These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA. Genes Cells, 2000 Oct, 5(10), 803 - 13 Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution; Yoshikawa M et al.; BACKGROUND: Crystallographic and mutational studies of Escherichia coli RuvC Holliday junction resolvase have revealed that a catalytic site of each subunit is composed of four acidic residues at the bottom of the putative DNA-binding cleft, whose surface contains eight basic residues . RESULTS: To elucidate the functional roles of the basic residues on the cleft surface, we constructed a series of mutant ruvC genes and characterized their properties in vivo and in vitro . Among them, two RuvC mutants with a single alteration, K107A and K118A, were defective in UV-repair and showed a dominant negative effect . The purified K107A and K118A proteins showed reduced binding activity to the junction DNA in the presence of Mg2+ under high salt conditions . Mn2+ increased both the junction binding and cleaving activities of the mutant proteins . In the absence of a divalent cation, the wild-type, K107A and K118A proteins did not bind to junction DNA under high salt conditions, but the D7N mutant, with an alteration of the catalytic centre, was able to bind to the junction efficiently . CONCLUSION: The results presented here, in conjunction with previous crystallographic studies, suggest that the catalytic complex which is formed through interactions of acidic residues, Mg2+ and a cleavable phosphodiester bond, is stabilized by Lys-107 and Lys-118 via electrostatic interactions with the DNA backbone, a process which is critically important for the cleavage reaction to take place . One or two basic residues near the catalytic centre have also been found in other RNase H superfamily proteins, indicating that this is the conserved reaction mechanism in this superfamily. Eur J Biochem, 2000 Nov, 267(21), 6470 - 5 Domain structure of tropomodulin: distinct properties of the N-terminal and C-terminal halves; Kostyukova A et al.; The structure of tropomodulin, the unique capping protein for the pointed end (the slow-growing end) of an actin filament, was studied . An improved Escherichia coli expression system for chicken E-tropomodulin was established and tropomodulin was prepared, Tmod (N39), in which 15 amino acid residues from the original C-terminus are deleted at the DNA level . This expression and purification system accidentally co-produces an 11-kDa fragment with the original N-terminus (N11) . By applying limited proteolysis to Tmod (N39), a 20-kDa C-terminal fragment (C20) was obtained . The limited proteolysis data, as well as the fluorescence spectrometry and CD analyses of Tmod (N39), C20 and N11, revealed that tropomodulin is an alpha-helical protein that consists of two distinct domains . The C-terminal half (20 kDa) is resistant to proteolysis, which suggests that this domain is tightly folded . In contrast, the N-terminal half is susceptible to proteolysis, indicating that in solution this half is likely to be extended or to form a highly flexible structure . Cross-linking experiments with glutaraldehyde indicated that Tmod (N39) and N11 can form complexes with tropomyosin, whereas C20 cannot . This confirms the previous report that the site(s) of interaction with tropomyosin resides in the N-terminal 11-kDa region of tropomodulin. Eur J Biochem, 2000 Nov, 267(21), 6369 - 77 Biochemical analysis of a thermostable tryptophan synthase from a hyperthermophilic archaeon; Tang XF et al.; Pyridoxal 5'-phosphate-dependent tryptophan synthase catalyzes the last two reactions of tryptophan biosynthesis, and is comprised of two distinct subunits, alpha and beta . TktrpA and TktrpB, which encode the alpha subunit and beta subunit of tryptophan synthase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, were independently expressed in Escherichia coli and their protein products were purified . Tryptophan synthase complex (Tk-TS complex), obtained by heat treatment of a mixture of the cell-free extracts containing each subunit, was also purified . Gel-filtration chromatography revealed that Tk-TrpA was a monomer (alpha), Tk-TrpB was a dimer (beta2), and Tk-TS complex was a tetramer (alpha2 beta2) . The Tk-TS complex catalyzed the overall alphabeta reaction with a specific activity of 110 micromol Trp per micromol active site per min under its optimal conditions (80 degrees C, pH 8.5) . Individual activity of the alpha and beta reactions of the Tk-TS complex were 8.5 micromol indole per micromol active site per min (70 degrees C, pH 7.0) and 119 micromol Trp per micromol active site per min (90 degrees C, pH 7.0), respectively . The low activity of the alpha reaction of the Tk-TS complex indicated that turnover of the beta reaction, namely the consumption of indole, was necessary for efficient progression of the alpha reaction . The alpha and beta reaction activities of independently purified Tk-TrpA and Tk-TrpB were 10-fold lower than the respective activities detected from the Tk-TS complex, indicating that during heat treatment, each subunit was necessary for the other to obtain a proper conformation for high enzyme activity . Tk-TrpA showed only trace activities at all temperatures examined (40-85 degrees C) . Tk-TrpB also displayed low levels of activity at temperatures below 70 degrees C . However, Tk-TrpB activity increased at temperatures above 70 degrees C, and eventually at 100 degrees C, reached an equivalent level of activity with the beta reaction activity of Tk-TS complex . Taking into account the results of circular dichroism analyses of the three enzymes, a model is proposed which explains the relationship between structure and activity of the alpha and beta subunits with changes in temperature . This is the first report of an archaeal tryptophan synthase, and the first biochemical analysis of a thermostable tryptophan synthase at high temperature. Eur J Biochem, 2000 Nov, 267(21), 6346 - 52 Identification of neoxanthin synthase as a carotenoid cyclase paralog; Bouvier F et al.; Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants . A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin-capsorubin synthase (CCS) . The identified tomato NSY cDNA (T.NSY) encodes a 56-kDa plastid-targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin . In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed . NSY is structurally similar to LCY and CCS . However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present . LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids. J Bacteriol, 2000 Nov, 182(21), 6247 - 9 Polyamine transport and role of potE in response to osmotic stress in Escherichia coli; Schiller D et al.; When transport of polyamines in Escherichia coli was examined, putrescine excretion was observed under two different physiological conditions: (i) strictly correlated to growth and (ii) following a hyperosmotic shock . Spermidine was not excreted . Characterization of a deletion mutant showed that PotE is not involved in these transport processes. J Bacteriol, 2000 Nov, 182(21), 6243 - 6 Identification of the ubiD gene on the Escherichia coli chromosome; Zhang H et al.; The open reading frame at 86.7 min on the Escherichia coli chromosome, "yigC," complemented a ubiD mutant strain, AN66, indicating that yigC is the ubiD gene . The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database. J Bacteriol, 2000 Nov, 182(21), 6203 - 13 Deletion of the min operon results in increased thermosensitivity of an ftsZ84 mutant and abnormal FtsZ ring assembly, placement, and disassembly; Yu XC et al.; To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation of ftsZ, ftsZ84, with DeltaminCDE, a deletion of the entire min locus . Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42 degrees C but not at or above 42 degrees C . Contrary to expectations, the double mutant was significantly more thermosensitive than the ftsZ84 single mutant . When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity . Interestingly, even at 22 degrees C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles . Of these, a large number were present only at one pole . These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole . Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias . Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84 . These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells . A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells . The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures . Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes. J Bacteriol, 2000 Nov, 182(21), 6049 - 54 Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid; Holland-Staley CA et al.; Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and the translational levels . To study the AdhE protein, the adhE(+) structural gene was cloned into expression vectors under the control of the lacZ and trp(c) promoters . Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive . Constitutive mutants (adhC) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated . When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions . Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys . Thus, adhC strains contain a promoter mutation and two mutations in the structural gene . The mutant structural gene from adhC strains was designated adhE* . Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys . Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions . These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E . coli is physiologically significant. J Bacteriol, 2000 Nov, 182(21), 6014 - 26 Incompatibility protein IncC and global regulator KorB interact in active partition of promiscuous plasmid RK2; Rosche TM et al.; Replication of the broad-host-range, IncPalpha plasmid RK2 requires two plasmid loci: trfA, the replication initiator gene, and oriV, the origin of replication . While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts . The product of the incC gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA . Here we show that IncC has the properties expected of a component of an active partition system . The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2 . This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for incC-mediated incompatibility is a KorB-binding site (O(B)) . We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins . Elevated expression of the incC and korB genes individually has no obvious effect on Escherichia coli cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target . A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in an incC-dependent manner . Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing . These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2. J Bacteriol, 2000 Nov, 182(21), 5954 - 61 Sequence changes in the ton box region of BtuB affect its transport activities and interaction with TonB protein; Cadieux N et al.; Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic protein TonB . The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB . The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate . Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate . Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport . The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB . This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once . The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined . Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space . TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles. Am J Respir Crit Care Med, 2000 Oct, 162(4 Pt 1), 1366 - 71 Acid instillation enhances the inflammatory response to subsequent lipopolysaccharide challenge in rats; Yamada H et al.; Aspiration of gastric contents is one of leading causes of the acute respiratory distress syndrome (ARDS) . The pathogenesis of acid aspiration-induced acute lung injury is well understood . Less clear is why patients who have suffered acid aspiration are susceptible to ARDS . We studied the effects of acid instillation on the inflammatory response to subsequent lipopolysaccharide (LPS) challenge in rats . Instillation of acid into the right lung worsened the pathology induced by LPS that was administered 24 h after acid instillation . This included worsened oxygenation, increased pulmonary edema, increased production of tumor necrosis factor-alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant, neutrophil accumulation and mobilization to the alveolar spaces, and nitric oxide (NO) production . Of interest, neutrophil mobilization, NO production, and protein permeability were also magnified in the left lung . These effects were attenuated by administration of the protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin AG556 . These data suggest that acid instillation primes the rat to enhance the inflammatory response to subsequent endotoxin challenge and that at least part of the augmented inflammatory response depends on PTK. J Comb Chem, 2000 Sep-Oct, 2(5), 461 - 6 Rapid identification of substrates for novel proteases using a combinatorial peptide library; Rosse G et al.; Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology . The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library . The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A) . We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library . The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies. Anal Chem, 2000 Oct 1, 72(19), 4689 - 93 Extended-range glucose sensor employing engineered glucose dehydrogenases; Yamazaki T et al.; An enzyme glucose sensor with an expanded dynamic range was constructed using a novel strategy . This strategy was based on a new concept of utilizing protein-engineered enzymes with a different Michaelis constant, which allows for the expanded dynamic range . We used the engineered Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) of which His775 was substituted for Asp which showed an increased Km value (25-fold) . We first constructed the composite colorimetric analytical system employing the wild-type PQQGDH and His775Asp and evaluated its dynamic range . The composite colorimetric analytical system was constructed and showed a wide dynamic range of 0.5-30 mM with less than +/-5% error . The composite colorimetric analytical system, an extended-range colorimetric analytical system, enabled the determination of the concentration of glucose over a 30-fold range that could not have been achieved using the single colorimetric analytical system . Furthermore, we have demonstrated the composite amperometric glucose sensor employing the combination of His775Asn and His775Asp . The extended-range glucose sensor acquired not only the expanded dynamic range (3-70 mM) that covered both dynamic ranges of the single enzyme sensors but also the narrower substrate specificity of glucose due to the inherited property of engineered enzymes. Mutat Res, 2000 Oct 31, 470(2), 177 - 88 Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho{2, 1-b}furan (R7000), in lacI transgenic mice; Quillardet P et al.; The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho{2,1-b}furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue) . The mutation frequency was determined in various organs of i.p . - treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant . It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria . The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus . The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E . coli . The difference between mouse and E . coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions . Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans. J Biol Chem, 2001 Jan 12, 276(2), 884 - 94 The RihA, RihB, and RihC ribonucleoside hydrolases of Escherichia coli . Substrate specificity, gene expression, and regulation; Petersen C et al.; Pyrimidine-requiring cdd mutants of Escherichia coli deficient in cytidine deaminase utilize cytidine as a pyrimidine source by an alternative pathway . This has been presumed to involve phosphorylation of cytidine to CMP by cytidine/uridine kinase and subsequent hydrolysis of CMP to cytosine and ribose 5-phosphate by a putative CMP hydrolase . Here we show that cytidine, in cdd strains, is converted directly to cytosine and ribose by a ribonucleoside hydrolase encoded by the previously uncharacterized gene ybeK, which we have renamed rihA . The RihA enzyme is homologous to the products of two unlinked genes, yeiK and yaaF, which have been renamed rihB and rihC, respectively . The RihB enzyme was shown to be a pyrimidine-specific ribonucleoside hydrolase like RihA, whereas RihC hydrolyzed both pyrimidine and purine ribonucleosides . The physiological function of the ribonucleoside hydrolases in wild-type E . coli strains is enigmatic, as their activities are paralleled by the phosphorolytic activities of the nucleoside phosphorylases, and a triple mutant lacking all three hydrolytic activities grew normally . Furthermore, enzyme assays and lacZ gene fusion analysis indicated that rihB was essentially silent unless activated by mutation, whereas rihA and rihC were poorly expressed in glucose medium due to catabolite repression. Biochem Biophys Res Commun, 2000 Oct 14, 277(1), 216 - 20 Requirement for the COOH-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane in Escherichia coli; Kim DW et al.; Aqualysin I from Thermus aquaticus YT-1 is an extracellular subtilisin-type serine protease . The protease is synthesized as a distinct precursor composed of four functional domains: an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence . The N-terminal pro-sequence is essential for the production of active aqualysin I while the C-terminal pro-sequence is required for extracellular secretion of aqualysin I . In an E . coli expression system, the function of C-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane was investigated . More than 60-70% of the total activity was detected in the cytoplasmic fraction in the deletion mutations of the C-terminal pro-sequence while less than 30% was found in this fraction in wild type . In addition, in vitro processing of aqualysin I precursors with these mutations to a mature form promptly occurred and the folding into active aqualysin I was rapid . These results suggest that the C-terminal pro-sequence, probably in conjunction with the signal sequence, facilitates the translocation of the precursor across the cytoplasmic membrane by preventing the precursor from taking on an active conformation . Biochem Biophys Res Commun, 2000 Oct 14, 277(1), 147 - 51 Tracing the origin of the RACTK1 K(+) channel; Ortega B et al.; Potassium secretion by the kidney is vital for the maintenance of K(+) homeostasis . RACTK1, a putative inwardly rectifying potassium channel cloned from cultured rabbit collecting duct cells, has been proposed to play a role in this process . However, the lack of homology with any other cloned potassium channel and the inability to reproduce the results across different laboratories has brought into question the existence of RACTK1 . Recently, it has been suggested that RACTK1 is a contamination from Escherichia coli . In this work we add conclusive evidence supporting the bacterial origin of RACTK1 . Using both genomic PCR and RT-PCR we were unable to detect RACTK1 in a number of mammalian species . In addition sequencing of RACTK1 cDNA confirmed a complete homology between RACTK1 and a region of E . coli genomic DNA . Finally, a hypothesis on how RACTK1 could have been generated from a contamination by E . coli genomic DNA is presented . Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 1240 - 7 The glycosyl phosphatidylinositol anchor of human T-cadherin binds lipoproteins; Niermann T et al.; T-cadherin (T-cad) is a Ca(2+)-dependent cell adhesion glycoprotein bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor . T-cad expressed on vascular smooth muscle cells (SMC) binds lipoproteins on blot . To analyze the molecular basis for the interaction of T-cad with lipoproteins we expressed recombinant human T-cad in HEK293 cells . Whereas membrane-bound T-cad from SMC and T-cad transfected HEK293 cells bind lipoproteins, T-cadherin proteins cleaved from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) do not . The lipoprotein-binding function is also lacking both for a recombinant human T-cad expressed in HEK293 cells without the GPI signal sequence, and for a human T-cad form expressed in Escherichia coli that contains the signal sequence for GPI attachment but is not modified with a GPI . We conclude that the GPI moiety of T-cadherin is necessary and sufficient to mediate lipoprotein binding . Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 1199 - 202 The H159A mutant of yeast enolase 1 has significant activity; Brewer JM et al.; The function of His159 in the enolase mechanism is disputed . Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli . They reported minimal (ca . 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate . We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast . Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity . Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations . It is possible that His159 is necessary for correct folding of the enzyme and that expression in E . coli leads to largely misfolded protein . Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 924 - 9 The slow-binding inhibition of cathepsin K by its propeptide; Billington CJ et al.; A peptide corresponding to the full-length proregion (amino acids 16-114) of human cathepsin K was expressed and purified from Escherichia coli . This recombinant propeptide was investigated for its ability to inhibit the activity of three cysteine proteinases: cathepsins K, L, and B . Kinetic studies showed the propeptide to be a potent slow-binding inhibitor of its parent enzyme with a K(i) = 2 . 61 nM at pH 6 . This inhibition was pH-dependent, with a decrease in pH from 6 to 4 leading to a concomitant increase in K(i) to 147 nM . The propeptide also inhibited cathepsin L with a K(i) = 26.1 nM at pH 6, but showed little inhibition of cathepsin B at concentrations up to 400 nM . Biochem Biophys Res Commun, 2000 Sep 24, 276(2), 762 - 6 EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding; LeLean JE et al.; The EPR spectra of NH(2)-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined . From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g approximately 8, 4, 2) . A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L . A rhombic high spin signal (g approximately 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome . Accompanying this change was the appearance of a signal at g = 1.98 . Conversely, an axial high spin signal was observed (g approximately 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4 . Biochem Biophys Res Commun, 2000 Sep 24, 276(2), 553 - 8 Alteration of rat dipeptidyl peptidase III by site-directed mutagenesis: cysteine(176) is a regulatory residue for the enzyme activity; Li YH et al.; To comprehend the importance of cysteine residues for the regulation of enzyme activity, we replaced each of seven cysteine residues in rat dipeptidyl peptidase III cDNA with alanine, glycine, or glutamic acid residue using site-directed mutagenesis . Each mutated cDNA was expressed in E . coli (BL21), and each expressed DPP III was purified to apparent homogeneity on SDS-polyacrylamide gel electrophoresis . Six of the mutant proteins had similar activity to that of the wild-type enzyme, whereas the activity of the Cys(176) --> Ala mutant enzyme was only 25-35% of that of the wild-type enzyme activity . This mutant enzyme was resistant against both PCMB and NEM . Furthermore, both Cys(176) --> Gly and Cys(176) --> Glu mutated enzymes showed no DPP III activity . These seven mutated enzymes contained significant amounts of zinc, as determined by atomic absorption spectrometry . The results indicate that Cys(176) is essential for the regulation of DPP III activity . J Surg Res, 2000 Oct, 93(2), 265 - 71 Role of platelet-activating factor in leukocyte-independent plasma extravasation and mast cell activation during endotoxemia; Walther A et al.; BACKGROUND: Independently from leukocyte adherence, endothelial factors and mast cell activation seems to promote microvascular permeability . Platelet-activating factor (PAF) has been shown to play a significant role in endotoxin-induced leukocyte adherence . The aim of our study was to investigate if there is also a role for PAF in mediating leukocyte-independent microvascular permeability changes and activation of mast cells during endotoxemia . Therefore, during endotoxemia microvascular permeability and mast cell activation were determined after inhibition of L-selectin-mediated leukocyte adherence by fucoidin and after inhibition of PAF effects by the PAF receptor antagonist BN52021 . MATERIALS AND METHODS: In male Wistar rats, red cell velocity (V(RBC)), venular wall shear rate, microvascular permeability, leukocyte adherence, and mast cell activation were determined in mesenteric postcapillary venules using intravital microscopy at baseline and 60 and 120 min after start of a continuous infusion of endotoxin (ETX; 2 mg/kg/h, Escherichia coli O26:B6) (ETX group) . Animals in the FUCO/ETX group received fucoidin (25 mg/kg body wt) in addition to the procedure described above . Animals in the FUCO/ETX/PAF-ANT group received fucoidin and the PAF receptor antagonist BN52021 (5 mg/kg body wt) prior to the continuous endotoxin infusion . Control animals (control group) received only equivalent volumes of NaCl 0.9% . RESULTS: There were no microhemodynamic and macrohemodynamic differences between groups . In all endotoxin-challenged groups macromolecular leakage and mast cell activity increased significantly, starting at 60 min . Both macromolecular leakage and mast cell activity were significantly higher in the FUCO/ETX group than in the FUCO/ETX/PAF-ANT group and control group . Differences in macromolecular leakage between groups were significant at 120 min . Differences in mast cell activity between groups were significant at 60 and 120 min . CONCLUSIONS: The results of our study demonstrate a leukocyte-independent plasma extravasation that can be inhibited by the PAF receptor antagonist BN52021, indicating the involvement of PAF in the pathophysiology of leukocyte-independent microvascular damage during early endotoxemia . Mast cell activity seems to precede leukocyte-independent macromolecular leakage . Prostate, 2000 Oct 1, 45(2), 149 - 57 Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer; O'Keefe DS et al.; BACKGROUND: Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases . In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation . Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer . Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned . We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E . coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells . METHODS: Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter . The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line . We then replaced the luciferase gene with the E . coli cytosine deaminase gene in the subclone that showed the most luciferase activity . The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines . The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared . RESULTS: The enhancer region originally isolated from the PSMA gene was approximately 2 kb . Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long . The IC(50) of 5-FC was similar in all cell lines tested (>10 mM) . However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection . CONCLUSIONS: Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease . Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12170 - 5 DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli; Khodursky AB et al.; We investigated the global changes in mRNA abundance in Escherichia coli elicited by various perturbations of tryptophan metabolism . To do so we printed DNA microarrays containing 95% of all annotated E . coli ORFs . We determined the expression profile that is predominantly dictated by the activity of the tryptophan repressor . Only three operons, trp, mtr, and aroH, exhibited appreciable expression changes consistent with this profile . The quantitative changes we observed in mRNA levels for the five genes of the trp operon were consistent within a factor of 2, with expectations based on established Trp protein levels . Several operons known to be regulated by the TyrR protein, aroF-tyrA, aroL, aroP, and aroG, were down-regulated on addition of tryptophan . TyrR can be activated by any one of the three aromatic amino acids . Only one operon, tnaAB, was significantly activated by the presence of tryptophan in the medium . We uncovered a plethora of likely indirect effects of changes in tryptophan metabolism on intracellular mRNA pools, most prominent of which was the sensitivity of arginine biosynthetic operons to tryptophan starvation. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12350 - 5 Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils; Cahoon EB et al.; We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant . Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy . This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E . coli to support the activities of the plant fatty acid desaturase . Based on this, a substrate-dependent selection system was devised with the use of the E . coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs . Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants . These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase . Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil . The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition. Biochemistry, 2000 Oct 17, 39(41), 12595 - 605 Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis; Bergkvist A et al.; Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E . coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping . Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet . The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine {Prasad, G . S., et al . (1999) Nat . Struct . Biol . 6, 1126-1131} and human heart {White, A . W., et al . (2000) Structure 8, 1-12} transhydrogenases . Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates . To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI) . All mutants except R425C showed altered NADP(H) binding and domain interaction properties . In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI . Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context. Biochemistry, 2000 Oct 17, 39(41), 12585 - 94 Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase; Werner RM et al.; The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site . We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions . A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates . From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction . The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect . A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine . Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation . However, in contrast to a recent proposal {Parikh, S . S., et al . (2000) Proc Natl . Acad . Sci . 94, 5083}, there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG. Biochemistry, 2000 Oct 17, 39(41), 12575 - 84 Hypermodified nucleosides in the anticodon of tRNALys stabilize a canonical U-turn structure; Sundaram M et al.; Modified nucleosides in the anticodon domain of Escherichia coli tRNA(Lys) are necessary for high-affinity codon recognition and reading frame maintenance . Human tRNA(Lys,3) is the specific primer for HIV-1 reverse transcriptase and also requires nucleoside modification for proper function . We now present NMR solution structures for the fully modified 17-nucleotide E . coli tRNA(Lys) anticodon stem-loop domain (ASL) . NMR data were also collected for several partially modified ASLs, revealing the contributions each modified nucleoside (mnm(5)s(2)U34, t(6)A37, and psi39) makes in transforming the disordered, unmodified tRNA ASL into the highly ordered native structure . The solution structure of the native ASL domain provides insight into longstanding questions regarding both wobble position modification and the nearly ubiquitous t(6)A37 found in tRNAs with an adjacent U at position 36 . Native tRNA(Lys) has a U-turn structure similar to the yeast tRNA(Phe) crystal structure, unlike previously proposed "unconventional" anticodon structures characterized by stable interactions between mnm(5)s(2)U-34 and t(6)A-37. Biophys Chem, 2000 Aug 30, 86(2-3), 95 - 108 Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains; Wenk M et al.; Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E . coli . HgammaS and BgammaS are found to be authentic according to their spectral and hydrodynamic properties . Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities . The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH . In spite of its exemplary two-domain structure, gammaS-crystallin does not show bimodal unfolding characteristics . In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS the opposite holds true . Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions . Over the whole pH range between 2.0 and 11.5, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-C) follow the two-state model . The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains . Obviously, interactions between the domains do not contribute significantly to the overall stability of gammaS-crystallin . In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions. Lipids, 2000 Sep, 35(9), 937 - 45 Current methods for the identification and quantitation of ceramides: an overview; Cremesti AE et al.; Ceramides are key compounds in the metabolism of sphingolipids and are emerging as important second messengers for various cellular processes including cell cycle arrest, differentiation, senescence, apoptosis, and others . Because of their important biological functions, exact analysis of their molecular species and concentrations is crucial for elucidating their function and metabolism . Toward this goal, several methods have been developed for the identification and quantitation of cellular ceramide levels . Methods have been developed utilizing thin-layer or high-performance liquid chromatography . Mass spectrometry also has become increasingly utilized . The Escherichia coli diacylglycerol kinase assay is one of the most frequently used techniques for ceramide quantitation . This review presents a current summary of methods used for the identification and quantitation of ceramides. Mutat Res, 2000 Sep 18, 452(2), 197 - 210 Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice; Shane BS et al.; The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations . Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed . Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment . We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice . A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups . While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not . The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007) . The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI . The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C<C:G transversions in the cII spectrum resulting in a significant difference (p<0.0001) between the spectra . Our initial data indicates that although cII is as sensitive to mutation induction as lacI, fewer sites are available for certain classes of mutations to be manifest resulting in an apparent lack in change in the mutation spectrum. FEBS Lett, 2000 Oct 6, 482(3), 261 - 4 Mutilation of RNA phage Qbeta virus-like particles: from icosahedrons to rods; Cielens I et al.; Icosahedral virus-like particles (VLPs) of RNA phage Qbeta are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between beta-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasi-six-fold symmetry contacts of the VLPs . In order to reduce the stability of Qbeta VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence . It led to production in Escherichia coli cells of aberrant rod-like Qbeta VLPs, along with normal icosahedral capsids . The length of the rod-like particles exceeded 4-30 times the diameter of icosahedral Qbeta VLPs. FEBS Lett, 2000 Oct 6, 482(3), 215 - 9 Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase; Arechaga I et al.; Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies . Here, we report that the abundant over-production of subunit b of E . coli F(1)F(o) ATP synthase in the mutant host strains E . coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies . Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b . The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step . Recombinant subunit b represented up to 80% of the protein content of the membranes . The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin. FEBS Lett, 2000 Oct 6, 482(3), 180 - 4 Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation; Xu H et al.; Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6 . At pH 7.6, a molecular mass of 179.5+/-2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)(6) . At pH 5.8, (RepA)(6) associates to form a dimer with a molecular mass of 366.2+/-4.1 kDa . Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)(6) at pH 5.8 and 7.6 . The dissociation constants, K(d), for the equilibrium binding of (dA)(30) to the (RepA)(6) dimer at pH 5.8 and to (RepA)(6) at pH 7.6 were determined at 10 degrees C in the presence of 0.5 mM ATPgammaS, 10 mM MgCl(2) and 60 mM NaCl as K(d5.8)=0.94+/-0.13 microM at pH 5.8 and K(d7 . 6)=25.4+/-6.4 microM at pH 7.6 . The stoichiometries, n, for the two complexes (dA)(30)/(RepA)(6) dimer and (dA)(30)/(RepA)(6) at pH 5.8 and 7.6 were calculated from the corresponding binding curves . At pH 5.8 one (dA)(30) molecule was bound per (RepA)(6) dimer, while at pH 7.6 one (dA)(30) molecule was bound to one (RepA)(6) . Binding curves were compatible with a single ssDNA binding site present on the (RepA)(6) dimer and on (RepA)(6), respectively, with no indication of cooperativity . (RepA)(6) tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding . In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)(6) dimer was observed both in the absence and presence of (dA)(30). FEMS Microbiol Lett, 2000 Oct 15, 191(2), 259 - 63 Localization of the Leptospira interrogans metF gene on the CII secondary chromosome; Bourhy P et al.; An open reading frame of 885 nucleotides was identified as the Leptospira interrogans metF gene . The deduced amino acid sequence (294 amino acids) showed similarities with Escherichia coli methylene tetrahydrofolate reductase (MetF or MTHFR) (33% identity) and with the N-terminal part of human MTHFR (33% identity) . The L . interrogans metF gene complements an E . coli metF mutant to prototrophy, suggesting the functionality of the folate branch converging to form methionine . In addition, the L . interrogans MetF was found to be thermolabile . The metF gene belonged to the CII secondary chromosome, in contrast to the previously isolated metY and metX genes, which have been localized to the CI chromosome of Leptospira sp. FEMS Microbiol Lett, 2000 Oct 15, 191(2), 169 - 75 Conserved cytoplasmic motifs that distinguish sub-groups of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family; Anderson MS et al.; WecA, MraY and WbcO are conserved members of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family involved in the assembly of bacterial cell walls, and catalyze reactions involving a membrane-associated polyprenol phosphate acceptor substrate and a cytoplasmically located UDP-D-amino sugar donor . MraY, WbcO and WecA purportedly utilize different UDP-sugars, although the molecular basis of this specificity is largely unknown . However, domain variations involved in specificity are predicted to occur on the cytoplasmic side of the membrane, adjacent to conserved domains involved in the mechanistic activity, and with access to the cytoplasmically located sugar nucleotides . Conserved C-terminal domains have been identified that satisfy these criteria . Topological analyses indicate that they form the highly basic, fifth cytoplasmic loop between transmembrane regions IX and X . Four diverse loops are apparent, for MraY, WecA, WbcO and RgpG, that uniquely characterize these sub-groups of the transferase family, and a correlation is evident with the known or implied UDP-sugar specificity. Nucleic Acids Res, 2000 Oct 15, 28(20), 3926 - 34 Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase; Yu E et al.; We have shown previously that CrhC is a unique member of the DEAD-box family of RNA helicases whose expression occurs specifically under conditions of cold stress . Here we show that recombinant His-tagged CrhC, purified from Escherichia coli, is an ATP-independent RNA binding protein possessing RNA-dependent ATPase activity which is stimulated most efficiently by rRNA and polysome preparations . RNA strand displacement assays indicate that CrhC possesses RNA unwinding activity that is adenosine nucleotide specific . Unwinding of partially duplexed RNA proceeds in the 5'-->3' but not the 3'-->5' direction using standard assay conditions . Immunoprecipitation and far-western analysis indicate that CrhC is a component of a multisubunit complex, interacting specifically with a 37 kDa polypeptide . We propose that CrhC unwinds cold-stabilized secondary structure in the 5'-UTR of RNA during cold stress. J Virol, 2000 Nov, 74(21), 10260 - 8 Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli; Joshi SM et al.; Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA . It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles . Thus, p10 plays a role in spherical particle formation . To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins . The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant . Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E . coli were examined in crude extracts by electron microscopy . Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure . While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination . We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions . Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low . Deletion of the N-terminal residue(s) abrogated assembly . Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein. J Virol, 2000 Nov, 74(21), 10081 - 95 The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus; Doorbar J et al.; Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers . The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus . This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm . It binds to RNA in vitro, including the major HPV16 late transcript (E1E4 . L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4 . E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines . In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli . Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif . Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis . The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation . Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis. J Virol, 2000 Nov, 74(21), 9946 - 52 RNA binding properties of bunyamwera virus nucleocapsid protein and selective binding to an element in the 5' terminus of the negative-sense S segment; Osborne JC et al.; The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N) . Previous studies have suggested that the encapsidation signal may reside within the 5' terminus of each segment . The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography . An RNA probe containing the 5'-terminal 32 and 3'-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays . On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding . RNA-N complexes were resistant to digestion with up to 1 microg of RNase A per ml . Competition assays with a variety of viral and nonviral RNAs identified a region within the 5' terminus of the BUN S segment for which N had a high preference for binding . This site may constitute the signal for initiation of encapsidation by N. J Biol Chem, 2001 Jan 5, 276(1), 677 - 85 The role of histidine 632 in catalysis by human topoisomerase I; Yang Z et al.; Based on the crystal structure of human topoisomerase I, we hypothesized that hydrogen bonding between the side chain of the highly conserved His(632) and one of the nonbridging oxygens of the scissile phosphate contributes to catalysis by stabilizing the transition state . This hypothesis has been tested by examining the effects of changing His(632) to glutamine, asparagine, alanine, and tryptophan . The change to glutamine reduced both the relaxation activity and single-turnover cleavage activity by approximately 100-fold, whereas the same change at three other conserved histidines (positions 222, 367, and 406) had no significant effect on the relaxation activity . The properties of the mutant protein containing asparagine instead of histidine at position 632 were similar to those of the glutamine mutant, whereas mutations to alanine or tryptophan reduced the activity by approximately 4 orders of magnitude . The reduction in activity for the mutants was not due to alterations in substrate binding affinities or changes in the cleavage specificities of the proteins . The above results for the glutamine mutation in conjunction with the similar effects of pH on the wild type and the H632Q mutant enzyme rule out the possibility that His(632) acts as a general acid to protonate the leaving 5'-oxygen during the cleavage reaction . Taken together, these data strongly support the hypothesis that the only role for His(632) is to stabilize the pentavalent transition state through hydrogen bonding to one of the nonbridging oxygens. J Biol Chem, 2001 Jan 5, 276(1), 232 - 43 Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase; Harmon FG et al.; We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein . Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3 . Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species . Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA . The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm . Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product . In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1) . Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes. J Biol Chem, 2001 Jan 5, 276(1), 92 - 8 Purification and characterization of pol kappa, a DNA polymerase encoded by the human DINB1 gene; Gerlach VL et al.; The Escherichia coli dinB gene encodes DNA polymerase (pol) IV, a protein involved in increasing spontaneous mutations in vivo . The protein-coding region of DINB1, the human ortholog of DNA pol IV, was fused to glutathione S-transferase and expressed in insect cells . The purified fusion protein was shown to be a template-directed DNA polymerase that we propose to designate pol kappa . Human pol kappa lacks detectable 3' --> 5' proofreading exonuclease activity and is not stimulated by recombinant human proliferating cell nuclear antigen in vitro . Between pH 6.5 and 8.5, human pol kappa possesses optimal activity at 37 degrees C over the pH range 6.5-7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mm . Either Mg(2+) or Mn(2+) can satisfy a metal cofactor requirement for pol kappa activity, with Mg(2+) being preferred . Human pol kappa is unable to bypass a cisplatin adduct in the template . However, pol kappa shows limited bypass of an 2-acetylaminofluorene lesion and can incorporate dCTP or dTTP across from this lesion, suggesting that the bypass is potentially mutagenic . These results are consistent with a model in which pol kappa acts as a specialized DNA polymerase whose possible role is to facilitate the replication of templates containing abnormal bases, or possessing structurally aberrant replication forks that inhibit normal DNA synthesis. Biochem J, 2000 Oct 15, 351 Pt 2, 469 - 76 Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria; Wang L et al.; The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs . Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library . The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal . In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix . A single 2.4 kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0 kb) transcript was also observed . There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA . Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified . All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2'-deoxyuridine, but with different kinetic efficiencies . A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2 . The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography . These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy. J Mol Biol, 2000 Oct 20, 303(2), 213 - 25 Towards understanding a molecular switch mechanism: thermodynamic and crystallographic studies of the signal transduction protein CheY; Sola M et al.; The signal transduction protein CheY displays an alpha/beta-parallel polypeptide folding, including a highly unstable helix alpha4 and a strongly charged active site . Helix alpha4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment . Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ . The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg(2+) needed for the protein activation . Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix alpha 4, both to increase its level of stability and to improve its packing against the protein core . The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix alpha 4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface . Pharmacol Res, 2000 Nov, 42(5), 429 - 33 2-Mercaptoethylamine, radioprotector, inhibits the induction of the oxidative stress-inducible (soi) gene by paraquat in Escherichia coli; Kim IG et al.; To demonstrate the superoxide radical (.O(2)(-)) -scavenging activity of 2-mercaptoethylamine (MEA), we investigated the induction of the oxidative stress-inducible (soi) gene fused lacZ gene (soi-28:: lacZ) by the use of paraquat as a source of.O(2)(-) . When MEA or cysteine was added to the cultures before paraquat treatment, soi gene induction by paraquat was significantly inhibited . However, a high quantity of ascorbic acid (5 mm) inhibited soi gene induction by paraquat far less than MEA or cysteine did . The induction of soi gene induction by MEA exhibited a dose-dependent manner in the range of over 0.2 mm . The antagonistic molecules on the radioprotective action of MEA, ascorbic acid and cysteine did not counteract MEA action on the inhibition of paraquat-mediated soi gene induction . To clarify that the MEA action on the inhibition of paraquat-mediated soi gene induction may be due, in part, to.O(2)(-)-scavenging activity, we investigated the ability of MEA to inhibit the nitroblue tetrazolium (NBT) reduction mediated by.O(2)(-)generated in the xanthine oxidase/hypoxanthine system in vitro . At concentrations above 1 mm, MEA effectively inhibited the NBT reduction in a concentration-dependent fashion . Our results demonstrated that MEA has an ability to scavenge.O(2)(-), and so protects against.O(2)(-)-mediated damage . Cytokine, 2000 Oct, 12(10), 1519 - 25 Production of a biologically active human interleukin 18 requires its prior synthesis as PRO-IL-18; Liu B et al.; Interleukin (IL-)18 is an activator of NK cells and a co-inducer of Th(1)cytokines, sharing structural features with the IL-1 family of proteins . Unlike most other cytokines, IL-18 and IL-1beta lack a signal peptide, have an all beta-pleated sheet structure and are synthesized as biologically inactive precursors (pro-IL-18 and pro-IL-1beta) . These precursors are cleaved by caspase-1 (IL-1beta-converting enzyme, ICE) to form the biologically active mature cytokines . Direct expression of mature recombinant human IL-18 in E . coli resulted in a partially active cytokine . We tested the possibility that correct folding of huIL-18 requires its prior synthesis as pro-IL-18 . Because caspase-1 is not readily available, we constructed an expression vector encoding human pro-IL-18 in which the caspase-1 cleavage site was mutated into a factor Xa site . To facilitate purification, the mutated pro-IL-18 cDNA was fused in frame to a glutathione-S-transferase (GST) coding sequence . The GST-pro-IL-18 fusion protein was expressed in E . coli, captured on glutathione agarose and mature human IL-18, exhibiting high biological activity was released upon cleavage with factor Xa . This result indicates that correct folding of huIL-18 occurs at the level of pro-IL-18 and provides a practical way to produce biologically active huIL-18 . Parasitology, 1995 Nov, 111 ( Pt 4), 477 - 83 A female-specific cDNA sequence of Schistosoma mansoni encoding a mucin-like protein that is expressed in the epithelial cells of the reproductive duct; Menrath M et al.; Seven cDNA clones of Schistosoma mansoni containing the C-terminal part of the deduced sequence of a mucin-like protein have been identified . The protein contains 28% threonines, 20% serines, and has a pI of 3.4 . On Northern blots of RNA of adult worms, the cDNA clones detect 2 transcripts of 1.65 and 4.2 kb which are expressed only in female worms . The tissue of gene expression, as revealed by in situ hybridization, is the epithelium surrounding the female reproduction duct proximal to its entrance into the ootype . Accumulation of N-glycosylation sites suggests that the protein, like other mucins, might form a protective layer, coating the lining of the duct . Regarding its acidic pI, we hypothesize a role in preventing premature egg-shell formation . This is the first female-specifically transcribed sequence, hitherto known in S . mansoni that is not expressed in the vitellaria. J Med Microbiol, 2000 Oct, 49(10), 905 - 10 Effects of interaction between Escherichia coli verotoxin and lipopolysaccharide on cytokine induction and lethality in mice; Suzuki K et al.; In Escherichia coli 0157 infections, verotoxins (VT) play a critical role in causing the disease, although other factors such as lipopolysaccharide (LPS) and inflammatory cytokines may affect the progression and course of the disease . The present study examined the roles of VT and LPS in induction of serum cytokines and lethality in mice . LD50 of VT2 (13 ng) was c . 10(4)-fold smaller than that of LPS (400 microg) . Although the lethal toxicity of these toxins was examined in several experimental conditions, such as VT2 (5, 10, 20, 40 ng/mouse) alone or in combination with LPS (100 microg/mouse) at various times (-2 days to +2 days), no evidence of synergy was observed . VT2 did not augment LPS-induced tumour necrosis factor-alpha (TNF-alpha) or interleukin-6 production, and conversely suppressed TNF-alpha production when it was injected 2 days before LPS challenge . The data failed to indicate either synergic or additive effects of VT and LPS on cytokine production or lethality in mice . In contrast, antagonistic interactions were clearly observed in cytokine production in certain conditions . The results suggested that these toxins may be co-operatively involvedin the pathology of VT-related diseases, but not through synergic interactions. Clin Exp Pharmacol Physiol, 2000 Oct, 27(10), 787 - 92 Inhibition of cytokine release by and cardiac effects of type IV phosphodiesterase inhibition in early, profound endotoxaemia in vivo; Tofovic SP et al.; 1 . In rats, inhibition of type IV phosphodiesterase (PDE4) attenuates acute renal failure and early (hours) mortality induced by high-dose endotoxin . Because it is unlikely that protection of renal function accounts for improved early survivability, most likely PDE4 inhibition exerts multiple beneficial effects in endotoxaemia and the purpose of the present study was to test this hypothesis . 2 . In study 1, we determined, in anaesthetized rats, the effects of endotoxin (30 mg/kg, i.v.) on cardiac performance parameters (heart rate (HR), ventricular peak systolic pressure (VPSP), maximum positive change in left ventricular pressure with respect to time (+dP/dt), maximum negative change in left ventricular pressure with respect to time (-dP/dtmax), ventricular end-diastolic pressure (VEDP), ventricular minimum diastolic pressure (VMDP) and HR-pressure product), plasma catecholamine levels, plasma renin activity (PRA) and plasma levels of inflammatory cytokines (tumour necrosis factor (TNF)-alpha and interleukin (IL)-lbeta) . 3 . In study 2, we determined, in anaesthetized rats, whether inhibition of PDE4 attenuates lipopolysaccharide (LPS)-induced changes in the aforementioned parameters of heart performance and neurohumoral status . We compared the changes in these parameters induced by endotoxaemia in animals treated with either RO 20-1724 (10 microg/kg per min; a selective PDE4 inhibitor) or its vehicle (DMSO; 1.35 microL/min) . 4 . At 90 min postadministration, endotoxin significantly increased HR and reduced -dP/dtmax and VEDP and caused a several-fold increase in plasma levels of TNF-alpha, IL-1beta, noradrenaline, adrenaline and PRA . RO20-1724 significantly blunted the endotoxin-induced reduction in -dP/dtmax and decreased endotoxin-induced increases in TNF-alpha and IL-1beta without significantly altering endotoxin-induced changes in HR, VEDP, catecholamine levels and PRA . 5 . Results from these studies indicate that, in addition to preserving renal function, PDE4 inhibition attenuates inflammatory cytokine release caused by high-dose endotoxin and may have protective effects on diastolic function in early profound endotoxaemia. J Biol Chem, 2000 Dec 29, 275(52), 40874 - 8 Intersubunit interactions in Plasmodium falciparum thioredoxin reductase; Krnajski Z et al.; The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin . It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors . Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage . There exist two classes of TrxRs: the high M(r) and the low M(r) proteins . The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins . The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase . However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus . These residues have been shown to be crucial for the reduction of thioredoxin . In this study we address the question whether the active site residues of P . falciparum TrxR are provided by one or both subunits . Differentially tagged wild-type and PfTrxR mutants were co-expressed in E . coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins . Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit . Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites . In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR. J Biol Chem, 2001 Jan 12, 276(2), 1353 - 60 Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol; Vogel S et al.; Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol . Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II . We have identified an intracellular retinol-binding protein in these tissues . The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue . It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family . In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse . Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm . Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind . The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III. J Biol Chem, 2001 Jan 5, 276(1), 225 - 31 Identification of UvrY as the cognate response regulator for the BarA sensor kinase in Escherichia coli; Pernestig AK et al.; BarA is a membrane-associated protein that belongs to a subclass of tripartite sensors of the two-component signal transduction system family . In this study, we report that UvrY is the cognate response regulator for BarA of Escherichia coli . This conclusion is based upon homologies with analogous two-component systems and demonstrated by both biochemical and genetic means . We show that the purified BarA protein is able to autophosphorylate when incubated with {gamma-(32)P}ATP but not with {alpha-(32)P}ATP or {gamma-(32)P}GTP . Phosphorylated BarA, in turn, acts as an efficient phosphoryl group donor to UvrY but not to the non-cognate response regulators ArcA, PhoB, or CpxR . The specificity of the transphosphorylation reaction is further supported by the fact that UvrY can receive the phosphoryl group from BarA-P but not from the non-cognate tripartite sensor ArcB-P or ATP . In addition, genetic evidence that BarA and UvrY mediate the same signal transduction pathway is provided by the finding that both uvrY and barA mutant strains exhibit the same hydrogen peroxide hypersensitive phenotype . These results provide the first biochemical evidence as well as genetic support for a link between BarA and UvrY, suggesting that the two proteins constitute a new two-component system for gene regulation in Escherichia coli. Virology, 2000 Oct 10, 276(1), 27 - 36 Characterization of a beta-1,3-glucanase encoded by chlorella virus PBCV-1; Sun L et al.; Sequence analysis of the 330-kb chlorella virus PBCV-1 genome revealed an open-reading frame, A94L, that encodes a protein with significant amino acid identity to Glycoside Hydrolase Family 16 beta-1,3-glucanases . The a94l gene was cloned and the protein was expressed as a GST-A94L fusion protein in Escherichia coli . The recombinant A94L protein hydrolyzed the beta-1,3-glucose polymer laminarin and had slightly less hydrolytic activity on beta-1,3-1, 4-glucose polymers, lichenan and barley beta-glucan . The recombinant enzyme had the highest activity at 65 degrees C and pH 8 . We predicted that the a94l-encoded beta-1,3-glucanase is involved in degrading the host cell wall either during virus release and/or is packaged in the virion particle and involved in virus entry . Therefore, we expected a94l to be expressed late in virus infection . However, contrary to expectations, both the a94l mRNA and the A94L protein appeared 15 min after PBCV-1 infection and disappeared 60- and 120-min p.i . postinfection, respectively, indicating that a94l is an early gene . Twenty-seven of 42 chlorella viruses contained the a94l gene . To our knowledge, this is the first report of a virus-encoded beta-1,3-glucanase . Virology, 2000 Oct 10, 276(1), 7 - 15 In vitro strand transfer from broken RNAs results in mismatch but not frameshift mutations; DeStefano JJ et al.; An in vitro system to compare the fidelity of strand transfers from truncated vs full-length RNAs was constructed . A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer . All RNAs were derived from the N-terminal portion of the alpha-lac gene . On full-length donors, transfers occurred when DNAs migrated to the acceptor prior to being completed on the donor . On donors that were truncated, most transfers occurred after DNAs reached the end of the donor . Transfer products were amplified by PCR and used to replace the corresponding region in a vector containing the alpha-lac gene . Transformed Escherichia coli were screened for alpha-complementation by blue-white phenotype analysis, with white colonies scored as those with errors in alpha-lac . These errors were derived from RT synthesis and strand transfer . The mutant colony frequency approximately doubled for transfer products derived from truncated donors (0.026+/-0.005 vs . 0.053+/-0.011 (three experiments +/- SD), for full-length vs . truncated, respectively) . The increases resulted from additional non-template-directed bases (mostly thymidines) added to the DNAs before transfer . Sequence analysis of DNAs synthesized on truncated donors showed that about 60% had additions (20/34); however, those without additions transferred at a much higher rate than those with . Transfer of the DNAs with additions always resulted in substitutions; no frameshifts were observed . Results are consistent with RT adding nontemplated nucleotides at template termini . Transfer and subsequent extension of these products is severely inhibited relative to products without additions . The potential relevance of these findings to retrovirus replication is discussed . Neuropeptides, 2000 Jun-Aug, 34(3-4), 187 - 92 Augmenting effect of methionine-enkephalin on interleukin-6 production by cytokine-stimulated murine macrophages; Kowalski J et al.; The effects of methionine-enkephalin on the production of interleukin-6 by activated peritoneal murine macrophages were studied . Macrophage were activated with interleukin-1beta or interferon-gamma in the presence or absence of graded concentrations of methionine-enkephalin . Methionine-enkephalin combined with interleukin-1beta or interferon-gamma caused an increase in IL-6 release from cultured macrophages . The opioid receptor antagonist naloxone did not change the stimulatory effect of methionine-enkephalin on IL-6 production by stimulated macrophages . Methionine-enkephalin added to the culture medium of resting macrophages increased IL-6 release from macrophages which were later induced with interleukin-1beta or interferon-gamma . The results of this study suggest that methionine-enkephalin can modulate the proinflammatory cytokine response by controlling, via non-opioid receptor mechanism, the production of IL-6 . J Mol Biol, 2000 Oct 13, 303(1), 93 - 110 Molecular enzymology of the EcoRV DNA-(Adenine-N (6))-methyltransferase: kinetics of DNA binding and bending, kinetic mechanism and linear diffusion of the enzyme on DNA; Gowher H et al.; The EcoRV DNA-(adenine-N(6))-methyltransferase recognizes GATATC sequences and modifies the first adenine residue within this site . We show here, that the enzyme binds to the DNA and the cofactor S-adenosylmethionine (AdoMet) in an ordered bi-bi fashion, with AdoMet being bound first . M.EcoRV binds DNA in a non-specific manner and the enzyme searches for its recognition site by linear diffusion with a range of approximately 1800 bp . During linear diffusion the enzyme continuously scans the DNA for the presence of recognition sites . Upon specific M.EcoRV-DNA complex formation a strong increase in the fluorescence of an oligonucleotide containing a 2-aminopurine base analogue at the GAT-2AP-TC position is observed which, most likely, is correlated with DNA bending . In contrast to the GAT-2AP-TC substrate, a G-2AP-TATC substrate in which the target base is replaced by 2-aminopurine does not show an increase in fluorescence upon M.EcoRV binding, demonstrating that 2-aminopurine is not a general tool to detect base flipping . Stopped-flow experiments show that DNA bending is a fast process with rate constants >10 s(-1) . In the presence of cofactor, the specific complex adopts a second conformation, in which the target sequence is more tightly contacted by the enzyme . M.EcoRV exists in an open and in a closed state that are in slow equilibrium . Closing the open state is a slow process (rate constant approximately 0.7 min(-1)) that limits the rate of DNA methylation under single turnover conditions . Product release requires opening of the closed complex which is very slow (rate constant approximately 0.05-0.1 min(-1)) and limits the rate of DNA methylation under multiple turnover conditions . M.EcoRV methylates DNA sequences containing more than one recognition sites in a distributive manner . Since the dissociation rate from non-specific DNA does not depend on the length of the DNA fragment, DNA dissociation does not preferentially occur at the ends of the DNA . Microbiology, 2000 Oct, 146 ( Pt 10), 2655 - 63 A mutation which affects both the specificity of PtsG sugar transport and the regulation of ptsG expression by Mlc in Escherichia coli; Plumbridge J; Normally glucosamine (GlcN) is not a substrate for EIICB(Glc) of the glucose phosphotransferase system (PTS), encoded by ptsG, but it is transported by the mannose (Man) PTS, encoded by manXYZ . A mutation, umgC, has been described in Escherichia coli which allows a strain mutated in the Man PTS to grow on GlcN . The umgC mutation was mapped to the ptsG region and was proposed to make ptsG expression constitutive . Transcription of ptsG is regulated by the repressor Mlc so that mutations in mlc enhance the expression of ptsG . An mlc mutation, however, is not sufficient to allow good growth on GlcN, unlike the umgC mutation . The umgC mutation is shown to enhance expression of ptsG even in the absence of any PTS sugar transport, but the increase is greater in the presence of GlcN or Man . The umgC mutation also increases expression of the ptsHI and manXYZ operons, which are both regulated by Mlc . The umgC mutation was sequenced and two mutations were found: one, G176D, within the IIC membrane domain and the second, E472K, within the soluble IIB domain of PtsG . The cloned UmgC allele shows the enhanced transport and regulatory characteristics of the chromosomal mutation . Analysis of the two mutations present individually on plasmids shows that the IIC mutation is responsible for both the effect on sugar specificity and regulation. Microbiology, 2000 Oct, 146 ( Pt 10), 2643 - 53 T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis; Sommer N et al.; The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters . The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt . T4 early transcription is a very active process, possibly out-competing host transcription . The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two . To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E . coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus . Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1 . The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E . coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase . Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase . The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase . To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42 . The latter positions introduced into the E . coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters. J Biomol Struct Dyn, 2000 Aug, 18(1), 127 - 36 Structure of glutamate decarboxylase and related PLP-enzymes: computer-graphical studies; Areshev AG et al.; Amino acid sequences of E . coli glutamate decarboxylase (GADa) and those of 36 GAD of different origin were compared by pairwise alignment using computer program CLUSTAL . GADalpha and plant enzymes showed 59.8-67.8% subunit homology, GADalpha and other bacterial GAD--49.8-77.6%, whereas GADalpha and animal enzymes--13.9-58.8% . Two PLP domains exhibited higher homology comparing to that of the whole subunit in the case of GAD67, plant (68.4-73.9%), and bacterial (46.7-83.2%) enzymes . The alignment of PLP-domains of 37 GAD, three group II decarboxylases, and two pyridoxal enzymes with known 3D structures (bacterial ORD and mAAT from chicken heart) allowed us to reveal conserved residues of the active sites . Their functional role is discussed . Modelling of the PLP-binding sites in active centers for GADalpha and human brain GAD67 was done using the Swiss-PdbViewer homology modelling program . Although the homology between GADalpha and GAD67 is rather low, structural similarity of their active sites allows us to consider here a functional convergence . Thus, glutamate decarboxylation by GADalpha may be helpful for understanding general mechanism of this reaction. J Appl Microbiol, 2000 Sep, 89(3), 390 - 6 A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking water; Van Poucke SO et al.; A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies . The test involves membrane filtration through a 25-mm black polyester filter, induction of beta-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-beta-Dglucuronide as an enzyme substrate and laser scanning of the membrane filter . Scan results can be confirmed on-line by epifluorescence microscopy . Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated > or =90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar . In 5.4% of all samples examined, SPC detected between 1 and 11 E . coli per 100 ml, while the two plate methods yielded negative results . Cases of a negative SPC result but a positive E . coli count on both reference media were not observed . This test would primarily be useful for 'emergency' monitoring of drinking water when rapid results are crucial. J Biol Chem, 2001 Jan 12, 276(2), 957 - 64 Excluded volume effects on the refolding and assembly of an oligomeric protein . GroEL, a case study; Galan A et al.; We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers . The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules . Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly . Crowding modulates GroEL oligomerization in two different ways . First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium . Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation . This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits . If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place . Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo. Methods, 2000 Oct, 22(2), 170 - 9 Low- and high-resolution mapping of DNA damage at specific sites; Li S et al.; Measurement of DNA damage and repair at the nucleotide level in intact cells has provided compelling evidence for the molecular details of these events as they occur in intact organisms . Furthermore, these measurements give the most accurate picture of the rates of repair in different structural domains of DNA in chromatin . In this report, we describe two methods currently used in our laboratories to map DNA lesions at (or near) nucleotide resolution in yeast cells . The low-resolution method couples damage-specific strand breaks in DNA with indirect end-labeling to measure DNA lesions over a span of 1.5 to 2 kb of DNA sequence . The resolution of this method is limited by the resolution of DNA length measurements on alkaline agarose gels (about +/-20 bp on average) . The high-resolution method uses streptavidin magnetic beads and special biotinylated oligonucleotides to facilitate end-labeling of DNA fragments specifically cleaved at damage sites . The latter method maps DNA damage sites at nucleotide resolution over a shorter distance (<500 bp), and is constrained to the length of DNA resolvable on DNA sequencing gels . These methods are used in tandem for answering questions regarding DNA damage and repair in different chromatin domains and states of gene expression . Methods, 2000 Oct, 22(2), 164 - 9 Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay; Kow YW et al.; Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites . Many procedures are available for the quantification and detection of base damage and AP sites . However, either these procedures are laborious or the starting materials are difficult to obtain . A biotinylated aldehyde-specific reagent, ARP, has been shown to react specifically with the aldehyde group present in AP sites, resulting in biotin-tagged AP sites in DNA . The biotin-tagged AP sites can then be determined colorimetrically with an ELISA-like assay, using avidin/biotin-conjugated horseradish peroxidase as the indicator enzyme . The ARP assay is thus a simple, rapid, and sensitive method for the detection of AP sites in DNA . Furthermore, removal of damaged base by DNA N-glycosylases generates AP sites that can be measured by the ARP reagent . By coupling the ARP assay with either endonuclease III from Escherichia coli or 8-oxoguanine N-glycosylase (OGG1) from yeast, investigators can rapidly determine the amount of oxidative pyrimidine damage (endonuclease III-sensitive sites) or purine damage (OGG1-sensitive sites) in cellular DNA, respectively . An increased level of oxidative damage has been implicated in several age-related human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as the aging process . The sensitivity and simplicity of the ARP assay thus make it a valuable method for investigators who are interested in estimating the level of oxidative DNA damage in cells and tissues derived from patients with various age-related diseases or cancers . Methods, 2000 Oct, 22(2), 148 - 56 Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology; Rodriguez H et al.; Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA . DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III . The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR) . In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR . Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA . Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2 . Methods, 2000 Oct, 22(2), 135 - 47 Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction; Ayala-Torres S et al.; Soon after discovery of the polymerase chain reaction (PCR), various laboratories have attempted to use quantitative PCR (QPCR) to detect DNA damage in specific gene segments . The development of techniques that facilitate long PCR increased the sensitivity of the assay so that biologically relevant doses of DNA-damaging agents could be assessed . QPCR has been used to survey DNA damage induced by different genotoxicants and to establish the repair kinetics of numerous genes . Current work seeks to analyze damage and repair in specific genes from animals exposed to specific DNA-damaging agents such as oxidative stress . Methods, 2000 Oct, 22(2), 120 - 6 In vivo technique for determining transcriptional mutagenesis; You HJ et al.; When an elongating RNA polymerase encounters DNA damage on the template strand of a transcribed gene it can either be arrested by or be transcribed through the lesion . Lesions that arrest RNA polymerases are thought to be subject to transcription-coupled repair, whereas that damage that is bypassed can cause miscoding, resulting in "mutations" in the transcript (transcriptional mutagenesis) . We have developed a technique using a plasmid-based luciferase reporter assay to determine the extent to which a particular type of DNA base modification is capable of causing transcriptional mutagenesis in vivo . The system uses Escherichia coli strains with different DNA repair backgrounds and is designed to detect phenotypic changes caused by transcriptional mutagenesis under nongrowth conditions . In addition, this method is capable of indicating the extent to which a particular DNA repair enzyme (or pathway) suppresses the occurrence of transcriptional mutagenesis . Thus, this technique provides a tool with which the effects of various genes on non-replication-dependent pathways resulting in the generation of mutant proteins can be gauged . Poult Sci, 2000 Sep, 79(9), 1271 - 5 Alloantigen systems L and P influence phagocytic function independent of the major histocompatability complex (B) in chickens; Qureshi MA et al.; Synthetic parent stocks were designed to produce progeny among which alleles were simultaneously segregating for nine alloantigen systems, including the MHC (B) . Chicks from Ancona-derived B19B19 females crossed with White leghorn B19B21 males were blood typed, resulting in genotypic categories for the A-E, C, D, H, I, L, and P loci with the objective of determining which, if any, of the eight non-MHC alloantigen systems influence or interact with the B system genotypes for blood monocyte phagocytic activity . Leukocytes obtained from whole blood at 2 and 4 wk were separated on a Fico/Lite LymphoH, density gradient and were allowed to adhere to glass coverslips . The resulting adherent monocyte monolayers were incubated with viable Escherichia coli for 1 h and stained with Leukostat, and the phagocytic monocytes and numbers of internalized bacteria per phagocytic monocyte were scored microscopically . The combined results from two separate trials demonstrated that the genotypes of the A-E, C, D, H, and I systems did not differ in the percentage of monocytes exhibiting phagocytosis, whereas significant differences were noted relative to the B system genotype at 2 wk of age (B19B21 > B19B19; P = 0.049), L at 4 wk (L1L1 > L1L2; P = 0.009), and P at 4 wk (P4P4 > P1P1; P = 0.047) . The data were further analyzed to determine any interactions of P and L alloantigen genotypes with the B system genotypes; no such interaction was observed . These studies suggest that the L and P non-MHC alloantigen systems have the potential to influence immune responses by modulating phagocytic function in chickens . Furthermore, this modulation seems to be independent of the B (MHC) system. Phys Rev Lett, 2000 Mar 27, 84(13), 3005 - 8 Thermodynamics of heat-shock response; Bourke Arnvig K et al.; Production of heat-shock proteins is induced when a living cell is exposed to a rise in temperature . The heat-shock response of protein DnaK synthesis in E.coli for temperature shifts T-->T+DeltaT and T-->T-DeltaT is measured as a function of the initial temperature T . We observe a reversed heat shock at low T . The magnitude of the shock increases when one increases the distance to the temperature T0 approximately 23 degrees C, thereby mimicking the nonmonotonous stability of proteins at low temperature . This suggests that stability related to hot as well as cold unfolding of proteins is directly implemented in the biological control of protein folding. Biochim Biophys Acta, 2000 Sep 29, 1481(2), 349 - 59 Dynamics of a mobile loop at the active site of Escherichia coli asparaginase; Aung HP et al.; Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases . Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles . Another common feature is a mobile surface loop that closes over the active site when the substrates is bound . We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe . With wild-type enzyme the fluorescence of the only tryptophan, W66, was monitored . Here asparagine induced a rapid closure of the loop . The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step . A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan . In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and >10(3), respectively, while substrate binding was unaffected . This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis . Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover. Biochim Biophys Acta, 2000 Sep 29, 1481(2), 297 - 309 Characterization of the ubiquitin-specific protease activity of the mouse/human Unp/Unph oncoprotein; Gilchrist CA et al.; The ubiquitin-specific proteases (Ubps) are a family of largely dissimilar enzymes with two major conserved sequence regions, containing either a conserved cysteine residue or two conserved histidine residues, respectively . The murine Unp oncoprotein and its human homologue, Unph, both contain regions similar to the conserved Cys and His boxes common to all the Ubps . In this study we show that Unp and Unph are active deubiquitinating enzymes, being able to cleave ubiquitin from both natural and engineered linear ubiquitin-protein fusions, including the polyubiquitin precursor . Mutation of the conserved Unp Cys and His residues abolishes this activity, and identifies the likely His residue in the catalytic triad . Unp is tumorigenic when overexpressed in mice, leading to the suggestion that Unp may play a role in the regulation of ubiquitin-dependent protein degradation . We have demonstrated here that the high-level expression of Unp in yeast does not disrupt the degradation of the N-end rule substrate Tyr-beta-galactosidase (betagal), the non-N-end rule substrate ubiquitin-Pro-betagal, or the degradation of abnormal, canavanine-containing proteins . These data suggest that Unp is not a general modulator of ubiquitin-dependent proteolysis . However, Unp may have a role in the regulation of the degradation of a specific, as yet undescribed, substrate(s). Biochim Biophys Acta, 2000 Sep 29, 1468(1-2), 367 - 80 The transport modifier RS1 is localized at the inner side of the plasma membrane and changes membrane capacitance; Valentin M et al.; Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2 . These effects were dependent on the species of RS1 and on the target transporters . hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types . Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1 . In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated . After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated . Also after overexpression in X . laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol . Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane . Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport . Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface . The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane . Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane. Biochim Biophys Acta, 2000 Sep 29, 1468(1-2), 175 - 86 Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids; White GF et al.; Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes . Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins . Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution . The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E . coli polar lipid by extrusion . Liposomes prepared from E . coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy . At high concentrations, NaCl caused aggregation of E . coli lipid liposomes that precluded detailed interpretation of light scattering data . Proteoliposomes and liposomes prepared from E . coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte . These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E . coli lipid vesicles. Mutat Res, 2000 Oct 16, 461(2), 145 - 56 Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity; Schulz I et al.; We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz . {4-(tert-butyldioxycarbonyl)benzyl}triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer . The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins . In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein . The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2 . In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23% . In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein . The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC-->TA and GC-->CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively . In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC-->CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins. FEBS Lett, 2000 Sep 29, 482(1-2), 19 - 24 CEO1, a new protein from Arabidopsis thaliana, protects yeast against oxidative damage; Belles-Boix E et al.; The Saccharomyces cerevisiae strain WYT, deficient in the YAP1 transcription factor, was used in a molecular screen to identify genes from Arabidopsis thaliana that could overcome the oxidative stress-sensitive phenotype of these yeast cells . A cDNA named CEO1 increased the tolerance to oxidative damage caused by tert-butylhydroperoxide of both the Yap1(-) mutant and the wild-type yeast . Additionally, in Yap1(-) yeast, CEO1 also induced cross-tolerance to oxidative damage caused by hydrogen peroxide and diamide . CEO1 was assigned as being part of a small gene family that, until now, is exclusively restricted to plants . In Arabidopsis, CEO1 was produced in all organs, especially in roots and stems . By using the yeast two-hybrid system, proteins that specifically interact with CEO1 in yeast were identified, and putative DNA-binding proteins were consistently recovered. Biochim Biophys Acta, 2000 Sep 27, 1487(2-3), 190 - 200 Augmented resistance to oxidative stress in fatty rat livers induced by a short-term sucrose-rich diet; Spolarics Z et al.; Hepatic steatosis and the accompanying oxidative stress have been associated with a variety of liver diseases . It is not known if fat accumulation per se plays a direct role in the oxidative stress of the organ . This study tested if steatosis induced by a short-term carbohydrate-rich diet results in an increased hepatic sensitivity to oxidative stress . Antioxidant status was determined in a liver perfusion system and in isolated parenchymal, endothelial and Kupffer cells from rats kept on sucrose-rich diet or on regular diet for 48 h . t-Butyl hydroperoxide addition (2 mM) to the perfusion fluid resulted in a release of alanine aminotransferase (ALT) in livers from controls, whereas no ALT release was observed in fatty livers . After t-butyl hydroperoxide addition, oxidized glutathione release was 40% less in fatty than in control livers, whereas reduced glutathione (GSH) release was not different . Sinusoidal oxidant stress was mimicked by the addition of lipopolysaccharide (LPS) from Escherichia coli (10 microg/ml) followed by the addition of opsonized zymosan (8 mg/ml) to the perfusion medium . LPS plus zymosan treatments resulted in the release of ALT in control but not in fatty livers . At the end of perfusion, liver glutathione content was 3-fold elevated, and the tissue content of lipid peroxidation products was approx . 40% less in fatty livers compared to controls . GSH content was doubled and glucose-6-phosphate dehydrogenase (G6PD) expression was elevated by 3- and 10-fold in sinusoidal endothelial and parenchymal cells form fatty livers compared to cells from control animals . Following H(2)O(2) administration in vitro (0.2-1 mM), GSH remained elevated in endothelial and parenchymal cells from fatty livers compared to cells from controls . In contrast, G6PD activity and GSH content were similar in Kupffer cells isolated from fatty or control livers . The study shows that hepatic fat accumulation caused by a short-term sucrose diet is not accompanied by elevated hepatic lipid peroxidation, and an elevated hepatic antioxidant activity can be manifested in the presence of prominent steatosis . The diet-induced increase in G6PD expression and, thus, the efficient maintenance of reduced glutathione in endothelial and parenchymal cells are a supportive mechanism in the observed hepatic resistance against intracellular or sinusoidal oxidative stress. Biochimie, 2000 Aug, 82(8), 705 - 15 Myristoylation-dependent N-terminal cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cellular extracts; Braun T et al.; The myristoylated alanine-rich C kinase substrate (MARCKS) has been proposed to regulate the plasticity of the actin cytoskeleton at its site of attachment to membranes . In macrophages, MARCKS is implicated in various cellular events including motility, adhesion and phagocytosis . In this report we show that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E . coli, between Lys-6 and Thr-7 . Cleavage of MARCKS decreases its affinity for macrophage membranes by ca . one order of magnitude, highlighting the contribution of the myristoyl moiety of MARCKS to membrane binding . Importantly, cleavage requires myristoylation of MARCKS . Furthermore, MARCKS-related protein (MRP), the second member of the MARCKS family, is not digested . Since Thr-7 is lacking in MRP this suggests that Thr-7 at the P1 position is important for the recognition of lipid-modified substrates . A different product is observed when MARCKS is incubated with a calf brain cytosolic extract . This product can be remyristoylated in the presence of myristoyl-CoA and N-myristoyl transferase, demonstrating that cycles of myristoylation/demyristoylation of MARCKS can be achieved in vitro . Although the physiological relevance of these enzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrates. Biochimie, 2000 Aug, 82(8), 693 - 704 Overproduction and improved strategies to purify the threenative forms of nuclease-free HU protein; Pellegrini O et al.; The heterodimeric HU protein was isolated from Escherichia coli as one of the most abundant DNA binding proteins associated with the bacterial nucleoid . HUalphabeta is composed of two very homologous subunits, but HU can also be present in E . coli under its two homodimeric forms, HUalpha(2) and HUbeta(2) . This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts and in some viruses . HU can participate, like the histones, in the maintenance of DNA supercoiling and in DNA condensation . This protein which does not recognize any specific sequence on double-stranded DNA, has been shown to bind specifically to cruciform DNA as does the eukaryotic HMG1 protein and to a series of structures which are found as intermediates of DNA repair, e.g., nick, gap, 3'overhang, etc . The strong binding of HU to these diverse DNA structures could explain, in part at least, its pleiotropic role in the bacterial cell . To understand all the facets of its interactions with nucleic acids, it was necessary to develop a procedure which allowed the purification of the three forms of HU under their native form and without the nuclease activity strongly associated with the protein . We describe here such a procedure as well as demonstrating that the three histidine-tagged HUs we have produced, have conserved the binding characteristics of native HUs . Interestingly, by two complementation tests, we show that the histidine-tagged HUs are fully active in vivo. Biochimie, 2000 Aug, 82(8), 683 - 91 A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1; Dahlgren A et al.; Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination . RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA . RF1 and RF2 have been shown to bind to several ribosomal proteins . To study this interaction in vivo, prfA1, a mutant form of RF1 has been used . A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 degrees C and shows an increased misreading of UAG and UAA . In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts(+); on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1 . The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive . The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16 . These three mutations all confer both a Ts (44 degrees C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not . The three alleles were sequenced and shown to be truncations of the S4 protein . None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation . Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants . Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough. Biochimie, 2000 Aug, 82(8), 671 - 82 Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination; Arkov AL et al.; Early studies provided evidence that peptide-chain release factors (RFs) bind to both ribosomal subunits and trigger translation termination . Although many ribosomal proteins have been implicated in termination, very few data present direct biochemical evidence for the involvement of rRNA . Particularly absent is direct evidence for a role of a large subunit rRNA in RF binding . Previously we demonstrated in vitro that mutations in Escherichia coli rRNAs, known to cause nonsense codon readthrough in vivo, reduce the efficiency of RF2-driven catalysis of peptidyl-tRNA hydrolysis . This reduction was consistent with the idea that in vivo defective termination at the mutant ribosomes contributes to the readthrough . Nevertheless, other explanations were also possible, because still missing was essential biochemical evidence for that idea, namely, decrease in productive association of RFs with the mutant ribosomes . Here we present such evidence using a new realistic in vitro termination assay . This study directly supports in vivo involvement in termination of conserved rRNA regions that also participate in other translational events . Furthermore, this study provides the first strong evidence for involvement of large subunit rRNA in RF binding, indicating that the same rRNA region interacts with factors that determine both elongation and termination of translation. Biochim Biophys Acta, 2000 Oct 2, 1493(3), 325 - 32 The DNA-binding domain of the gene regulatory protein AreA extends beyond the minimal zinc-finger region conserved between GATA proteins; Manfield IW et al.; The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif . A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a . at the C-terminus, and a much larger 728 a.a . sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E . coli and purified . The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis . The 91 a.a . protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a . protein has very low ( approximately microM) affinity for the same sequence . The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a . sequence C-terminal to the zinc finger . Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein . These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA . We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91). Annu Rev Microbiol, 2000, 54, 775 - 98 Proteins shared by the transcription and translation machines; Squires CL et al.; It is becoming increasingly clear that the complex machines involved in transcription and translation, the two major activities leading to gene expression, communicate directly with one another by sharing proteins . For some proteins, such as ribosomal proteins S10 and L4, there is strong evidence of their participation in both processes, and much is known about their role in both activities . The exact roles and interactions of other proteins, such as Nus factors B and G, in both transcription and translation remain a mystery . Although there are not, at present, many examples of such shared proteins, the importance of understanding their behavior and intimate involvement with two major cellular machines is beginning to be appreciated . Studies related to the dual activities of these proteins and searches for more examples of proteins shared between the transcription and translation machines should lead to a better understanding of the communication between these two activities and the purposes it serves. Annu Rev Microbiol, 2000, 54, 499 - 518 Functional modulation of Escherichia coli RNA polymerase; Ishihama A; The promoter recognition specificity of Escherichia coli RNA polymerase is modulated by replacement of the sigma subunit in the first step and by interaction with transcription factors in the second step . The overall differentiated state of approximately 2000 molecules of the RNA polymerase in a single cell can be estimated after measurement of both the intracellular concentrations and the RNA polymerase-binding affinities for all seven species of the sigma subunit and 100-150 transcription factors . The anticipated impact from this line of systematic approach is that the prediction of the expression hierarchy of approximately 4000 genes on the E . coli genome can be estimated. Fungal Genet Biol, 2000 Jul, 30(2), 147 - 53 A single five-step desaturase is involved in the carotenoid biosynthesis pathway to beta-carotene and torulene in Neurospora crassa; Hausmann A et al.; Phytoene desaturase Al-1 from Neurospora crassa was expressed in Escherichia coli and an active enzyme was isolated which catalyzed the stepwise introduction of up to five double bonds into the substrate phytoene . The major reaction products were 3, 4-didehydrolycopene and lycopene . Several of the desaturation intermediates, zeta-carotene, neurosporene, and lycopene, were also accepted as a substrate by Al-1 . In contrast to the structurally related bacterial enzymes, the cofactor involved in the dehydrogenation reaction was NAD for Al-1 . In situ competition with a neurosporene- and lycopene-converting hydratase and cyclase indicated that these enzymes can divert intermediates of the desaturation sequence . Based on the in vitro and in vivo results, the organization of the phytoene desaturase from N . crassa was proposed as an assembly of identical protein units which are responsible for the multistep reaction . However, the spatial arrangement should be loose enough to allow an exchange of individual intermediates in both directions in and out of this complex . Since gamma-carotene is not accepted as a substrate by Al-1, the formation of torulene must proceed exclusively by the cyclization of 3,4-didehydrolycopene . Nat Struct Biol, 2000 Oct, 7(10), 910 - 7 The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site; Abramson J et al.; Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases . These membrane proteins utilize different electron donors through dissimilar access mechanisms . We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli . The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase . Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site . A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities . In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase. Nat Struct Biol, 2000 Oct, 7(10), 876 - 80 Structure of the CO sensing transcription activator CooA; Lanzilotta WN et al.; CooA is a homodimeric transcription factor that belongs to the catabolite activator protein (CAP) family . Binding of CO to the heme groups of CooA leads to the transcription of genes involved in CO oxidation in Rhodospirillum rubrum . The 2.6 A structure of reduced (Fe2+) CooA reveals that His 77 in both subunits provides one heme ligand while the N-terminal nitrogen of Pro 2 from the opposite subunit provides the other ligand . A structural comparison of CooA in the absence of effector and DNA (off state) with that of CAP in the effector and DNA bound state (on state) leads to a plausible model for the mechanism of allosteric control in this class of proteins as well as the CO dependent activation of CooA. J Biol Chem, 2000 Dec 22, 275(51), 40142 - 7 K+ and ionic strength directly influence the autophosphorylation activity of the putative turgor sensor KdpD of Escherichia coli; Jung K et al.; The membrane-bound histidine kinase KdpD is a putative turgor sensor that regulates, together with the response regulator KdpE, the expression of the kdpFABC operon coding for the high affinity K(+)-uptake system KdpFABC of Escherichia coli . To elucidate the nature of the primary stimulus for KdpD, we developed an in vitro assay based on right-side-out membrane vesicles . Conditions were varied inside and outside of the vesicles, and KdpD autophosphorylation activity was tested . It was shown that an increase of the ionic strength inside the vesicles was accompanied by an increase of the autophosphorylation activity of KdpD with ATP . However, K(+) at concentrations higher than 1 mm inhibited KdpD autophosphorylation activity . This K(+)-specific effect was not observed with KdpD-Arg-511 --> Gln, a KdpD derivative, which causes K(+)-independent kdpFABC expression . When the osmolality outside the vesicles was increased, autophosphorylation activity of KdpD was stimulated, whereby salts were more effective than sugars . Treatment of the vesicles with amphipathic compounds did not affect KdpD autophosphorylation activity . Based on these results it is proposed that changes of intracellular parameters elicited by K(+) limitation or osmotic upshock directly influence KdpD autophosphorylation activity, whereby K(+) has an inhibitory and ionic strength a stimulatory effect. J Biol Chem, 2000 Dec 29, 275(52), 40933 - 7 Sodium channel activity in leukemia cells is directly controlled by actin polymerization; Negulyaev YA et al.; The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells . However, the molecular mechanisms underlying the changes in channel function remain to be defined . In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells . We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance . Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 microm {Ca(2+)}(i) . The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mm MgCl(2) to induce actin polymerization . In the absence of MgCl(2), addition of intact actin did not abolish the channel activity . Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32 . Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization . Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin. Mol Gen Genet, 2000 Sep, 264(1-2), 204 - 11 Cloning and characterization of a Legionella pneumophila-specific gene encoding a member of the LysR family of transcriptional regulators; Heuner K et al.; Flagellin gene regulation in Legionella pneumophila is modulated by various environmental factors . The expression of the virulent phenotype seems to be linked genetically to flagellum expression . To better understand the mechanisms of flagellin gene expression in L . pneumophila (Lp), we screened a pool of plasmids from a L . pneumophila Corby genomic library for the ability to prevent or reduce luciferase activity in the Escherichia coli strain YK410, which harbours a Lp-pflaA-luxAB fusion . We cloned a DNA fragment encoding the N-terminal part of a protein with significant similarity to members of the LysR family of transcriptional regulators (LTTRs) . The entire gene, cloned by inverse PCR, was named flaR . It encodes a protein of 302 amino acids, and computer-assisted analysis of the amino acid sequence revealed a helix-turn-helix motif located near the N-terminus of the protein . The FlaR protein exhibits 21-31% identity to various LTTRs . Furthermore, gel retardation experiments indicate that the FlaR protein is able to bind to its own promoter region and, to a lesser extent, to the flaA promoter of L . pneumophila . The flaR promoter region contains putative LysR binding motifs and two putative Fur boxes . Taken together, these results indicate that FlaR is a DNA-binding protein which belongs to the LTTR family . Southern analysis with a L . pneumophila Corby-specific flaR probe revealed homologous genes in various L . pneumophila strains, but not in the 12 nonpneumophila strains tested so far. Antonie Van Leeuwenhoek, 2000 Jul, 78(1), 23 - 31 Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress; Lindqvist A et al.; Isogenic strains of Escherichia coli that were defective in either of the two major aerobic terminal respiratory oxidases (cytochromes bo' and bd) or in the putative third oxidase (cytochrome bd-II) were studied to elucidate role(s) for oxidases in protecting cells from oxidative stress in the form of H2O2 and paraquat . Exponential phase cultures of all three oxidase mutants exhibited a greater decline in cell viability when exposed to H2O2 stress compared to the isogenic parent wild-type strain . Cytochrome bo' mutants showed the greatest sensitivity to H2O2 under all conditions studied indicating that this oxidase was crucial for protection from H2O2 in E . coli . Cell killing of all oxidase mutants by H2O2 was by an uncharacterized mechanism (mode 2 killing) with cell growth rate affected . The expression of phi(katG-lacZ), an indicator of intracellular H2O2, was 2-fold higher in a cydAB::kan mutant compared to the wild-type strain at low H2O2 concentrations (< 100 microM) suggesting that cytochrome bd mutants were experiencing higher intracellular levels of H2O2 . Protein fusions to the three oxidase genes demonstrated that expression of genes encoding cytochrome bd, but not cytochrome bo' or cytochrome bd-II was increased in the presence of external H2O2 . This increase in expression of 4P(cydA-lacZ) by H2O2 was further enhanced in a cyo::kan mutant . The level of cytochrome bd determined spectrally and phi(cydA-lacZ) expression was 5-fold and 2-fold higher respectively in an rpoS mutant compared to isogenic wild-type cells suggesting that RpoS was a negative regulator of cytochrome bd . Whether the effect of RpoS is direct or indirect remains to be determined. Cancer Res, 2000 Sep 15, 60(18), 5080 - 6 Increased mutant frequency and altered mutation spectrum of the lacI transgene in Wilson disease rats with hepatitis; Sone H et al.; The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver because of a mutation in the Atp7b gene, encoding a copper-ATPase, is a model of Wilson disease . It spontaneously develops hepatitis, and subsequently hepatocellular carcinoma and cholangiofibrosis . Excess intracellular copper has been thought to induce DNA damage through reactive oxygen species produced by Cu (II)/Cu (I) redox cycling, and also by direct interaction with DNA . We have developed lacI transgenic Wilson disease (WND-B) rats by mating LEC with Big Blue F344 rats carrying a lambda shuttle vector harboring the lacI gene . lacI mutations of the livers of C-B heterozygous (Atp7b w/m, lacI) and WND-B homozygous (Atp7b m/m, lacI) rats at 6, 24, and 40 weeks of ages were analyzed . Mutant frequencies in the WND-B rats were 2.0 +/- 0.7 x 10(-5), 5.3 +/- 0.9 x 10(-5), and 5.3 +/- 1.0 x 10(-5), respectively, significantly higher than those of C-B rats . Nucleotide sequence analysis revealed that the frequency of deletion mutations of more than two nucleotides were much higher, 15% in WND-B rats, but only 2% in C-B rats . In addition, the average size of deletion was larger in the former . Loss of oligonucleotide-repeat units was specific and relatively frequent in WND-B rats . This type of mutation might be implicated in the induction of DNA strand scissions by reactive oxygen species . These findings suggest that the increase in mutant frequencies and/or the specific type of mutation according to copper accumulation play a crucial role in hepatocarcinogenesis in LEC rats. Diabetes, 2000 Oct, 49(10), 1627 - 34 Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase; Delpierre G et al.; Fructosamines are thought to play an important role in the development of diabetic complications . Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates . The purpose of the present work was to identify and characterize the enzyme responsible for this conversion . Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine . The enzyme responsible for this conversion was purified approximately 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M(r) protein . Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs . Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes . Both proteins were expressed in Escherichia coli and purified . They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity . They also phosphorylated glycated lysozyme, though not unmodified lysozyme . Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety . The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins. J Clin Microbiol, 2000 Oct, 38(10), 3561 - 71 Serodiagnosis of Louse-Borne relapsing fever with glycerophosphodiester phosphodiesterase (GlpQ) from Borrelia recurrentis; Porcella SF et al.; Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality . Isolates of the causative agent, Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan . The glpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes . Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease . In the present work, we cloned and expressed the glpQ gene from B . recurrentis and used recombinant GlpQ in serological tests . Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of B . recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ . The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively . The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ . Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient . We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence. Biochemistry, 2000 Oct 10, 39(40), 12441 - 9 Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: extended X-ray absorption fine structure evidence for a metal-binding domain; Buchko GW et al.; Formamidopyrimidine-DNA glycosylase (Fpg) is a 30.2 kDa protein that plays an important role in the base excision repair of oxidatively damaged DNA in Escherichia coli . Sequence analysis and genetic evidence suggest that zinc is associated with a C4-type motif, C(244)-X(2)-C(247)-X(16)-C(264)-X(2)-C(267), located at the C-terminus of the protein . The zinc-associated motif has been shown to be essential for damaged DNA recognition . Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc-associated protein (ZnFpg) in the lyophilized state and in 10% frozen aqueous glycerol solution show directly that the metal is coordinated to the sulfur atom of four cysteine residues . The average Zn-S bond length is 2.33 +/- 0.01 and 2.34 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution . Fpg was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily cobalt-associated (CoFpg) . The profiles of the circular dichroism spectra for CoFpg and ZnFpg are identical, suggesting that the substitution of Co(2+) for Zn(2+) does not alter the structure of Fpg . A similar conclusion is reached upon the analysis of two-dimensional (15)N/(1)H HSQC spectra of uniformly (15)N-labeled samples of ZnFpg and CoFpg; the spectra are similar and display features characteristic of a structured protein . Biochemical assays with a 54 nt DNA oligomer containing 7, 8-dihydro-8-oxoguanine at a specific location show that CoFpg and ZnFpg are equally active at cleaving the DNA at the site of the oxidized guanine . EXAFS spectra of CoFpg indicate that the cobalt is coordinated to the sulfur atom of four cysteine residues with an average Co-S bond length of 2.28 +/- 0.01 and 2.29 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution . The structural similarity between CoFpg and ZnFpg suggests that it is biologically relevant to use the paramagnetic properties of Co(2+) as a structural probe. Biochemistry, 2000 Oct 10, 39(40), 12415 - 23 Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase; Chen L et al.; A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli . The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps . The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa) . At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively . Use of (D)-{5-(13)C}glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate . This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable . However, the most unique feature of A . fulgidus IPS was that it absolutely required divalent metal ions for activity . Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme . These properties suggested that this archaeal IPS was a class II aldolase . In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate. Biochemistry, 2000 Oct 10, 39(40), 12252 - 61 Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures; Hoare S et al.; The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies . The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene {(-)-anti-BPDE} by cis-covalent addition to N(2)-2'-deoxyguanosine {(-)-cis-anti-BP-N(2)-dG}, (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene {(+)-anti-5-MeCDE} by trans addition to N(2)-2'-deoxyguanosine {(+)-trans-anti-MC-N(2)-dG}, and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP) . The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated . The binding affinities of UvrA varied among the three adducts . UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM) . The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct . 5' Incisions occurred at the eighth phosphate from the modified guanine . The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts . However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds . Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts . Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates . The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates . A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it . These findings are discussed in terms of the available NMR solution structures. Biochemistry, 2000 Oct 10, 39(40), 12216 - 24 Mapping the functional domains of elongation factor-2 kinase; Pavur KS et al.; A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified {Futey, L . M., et al . (1995) J . Biol . Chem . 270, 523-529; Ryazanov, A . G., et al . (1997) Proc . Natl . Acad . Sci . U.S.A . 94, 4884-4889} . This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized {Ryazanov, A . G., et al . (1999) Curr . Biol . 9, R43-R45} . eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation . eEF-2 was the only known substrate for eEF-2 kinase . We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains . This enabled us to develop a rapid assay for eEF-2 kinase activity . To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis . We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1 . Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1 . Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1 . The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions . In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay . These results suggest that eEF-2 kinase consists of two domains connected by a linker region . The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain . The calmodulin-binding region is located between amino acids 51 and 96 . The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36". Biochemistry, 2000 Oct 10, 39(40), 12131 - 9 Folded state of the integral membrane colicin E1 immunity protein in solvents of mixed polarity; Taylor RM et al.; The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1 . Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system . Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3 . Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure . The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains . It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions . Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer region of the three transmembrane helices could contribute to the near-UV CD spectrum through helix-helix interactions. Genetics, 2000 Oct, 156(2), 477 - 88 Long-term experimental evolution in Escherichia coli . IX . Characterization of insertion sequence-mediated mutations and rearrangements; Schneider D et al.; As part of a long-term evolution experiment, two populations of Escherichia coli B adapted to a glucose minimal medium for 10,000 generations . In both populations, multiple IS-associated mutations arose that then went to fixation . We identify the affected genetic loci and characterize the molecular events that produced nine of these mutations . All nine were IS-mediated events, including simple insertions as well as recombination between homologous elements that generated inversions and deletions . Sequencing DNA adjacent to the insertions indicates that the affected genes are involved in central metabolism (knockouts of pykF and nadR), cell wall synthesis (adjacent to the promoter of pbpA-rodA), and ill-defined functions (knockouts of hokB-sokB and yfcU) . These genes are candidates for manipulation and competition experiments to determine whether the mutations were beneficial or merely hitchhiked to fixation. J Biomol NMR, 2000 Aug, 17(4), 311 - 22 Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli; Sorkin DL et al.; We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli . Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' delta1 protons near 100 ppm, and beta and alpha protons collectively between 20 and 50 ppm, in Fe(III)SOD . In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically . A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the beta protons of Asp 156, superseding earlier assignments (Ming et al . (1994) Inorg . Chem., 33, 83-87) . We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of 82 and epsilon1 protons . These are the closest protein protons to Fe(II) and therefore they have the broadest (approximately 4,000 Hz) and most difficult to detect resonances . Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state . Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c' (Bertini et al . (1988) Inorg . Chem., 37, 4814-4821 ) suggesting that Fe(II)SOD's equatorial His2Asp- ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+. J Biomol NMR, 2000 Aug, 17(4), 295 - 304 The auto-orientation in high magnetic fields of oxidized cytochrome b562 as source of constraints for solution structure determination; Arnesano F et al.; 15N-1H 1J couplings were measured at 500 MHz and 800 MHz for 15N enriched oxidized cytochrome b562 from E . coli . The magnetic field dependence of 70 1J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc) . The rdc were used as further constraints to improve the existing structure {Arnesano et al . (1999) Biochemistry, 38, 8657-8670} with a protocol which uses the rhombic anisotropy {Banci et al . (1998) J . Am . Ctherz . Soc., 120, 12903-12909} . The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions. J Am Soc Mass Spectrom, 2000 Oct, 11(10), 854 - 65 Matrix-assisted laser desorption/ionization mass spectrometry methods for oligodeoxynucleotides: improvements in matrix, detection limits, quantification, and sequencing; Zhang LK et al.; A comatrix of anthranilic acid and nicotinic acid is optimum for the matrix-assisted laser desorption/ionization time of flight determination of oligodeoxynucleotides that are comprised of up to 21 nucleotides . A detection limit of approximately 200 amol was obtained for an oligonucleotide 21mer . The comatrix system is also suitable for quantification of oligodeoxynucleotides provided an internal standard having one more or less nucleotide than the number in the analyte is used . Furthermore, the matrix, when used in combination with the ladder method of sequencing, allows the complete sequence of tens of picomoles of model oligodeoxynucleotides to be determined. Neurosurgery, 2000 Oct, 47(4), 931 - 8; discussion 938-9 Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma; Chang JW et al.; OBJECTIVE: In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model . METHODS: Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the beta-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-D-galactoside . CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis . For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-deltaE1 (as a control) . After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection . For in vivo antitumor experiments, 1x10(5) cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains . At 3 days after implantation, 1x10(8) plaque-forming units of ad-CD/TK or ad-deltaE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered . Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope . RESULTS: C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more . In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK . Obvious cytotoxicity (>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30 microg/ml or GCV at concentrations greater than 0.3 microg/ml at a multiplicity of infection of 100 . Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments . In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1+/-4.6 and 57.4+/-7.1 mm3, respectively) were significantly smaller than that of the control rats (157+/-8.9 mm3, P<0.05) . Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7+/-1.8 mm3 . CONCLUSION: These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene . Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors. Ugeskr Laeger, 2000 Sep 18, 162(38), 5074 - 7 {Dengue fever among 44 Danish travellers investigated in the department of epidemics at the Rigshospitalet during 1988-1998}; David KP et al.; Dengue fever is a major cause of febrile illness in the tropics, and we describe 44 patients with dengue fever seen at Rigshospitalet, Copenhagen from 1988-98 . A worldwide increase in transmission of dengue fever was reflected in the number of patients seen in 1997-1998 . All patients had fever and headache, and were biochemically characterised by thrombocytopenia, leucopenia, increased levels of alanine aminotransferase and a rise in haematocrit . One patient had dengue haemorrhagic fever, and two patients exhibited unusual reactions to the infection, one in the form of extended febrile neutropenia complicated by an episode of of E . coli septicaemia, and one patient developed progressive paralysis of both legs, and remains partially paretic. Anal Chem, 1998 Aug 1, 70(15), 3333 - 6 Collision-induced dissociation spectra obtained by Fourier transform ion cyclotron resonance mass spectrometry using a 13C,15N-doubly depleted protein; Akashi S et al.; Fourier transform ion cyclotron resonance mass spectra of 13C,15N-doubly depleted cystatin A M65L, produced by Escherichia coli grown on 99.9% {12C}glucose and 99.99% {14N}ammonium sulfate, showed salient monoisotopic peaks composed of 12C and 14N . Collision-induced dissociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trapping time in a radio frequency-only hexapole ion guide . Fragment ions in the spectra could be readily assigned to the amino acid sequence, owing to their markedly improved resolution and sensitivity as compared to those with the natural isotopic composition . Detailed analyses of the fragmentation patterns, facilitated by the use of 13C,15N-doubly depleted proteins, enabled the assignment of approximately 180 fragment ions to the sequence, while natural isotopic cystatin A allowed the assignment of approximately 110 fragment ions . Interestingly, no fragmentation was detected between residues 50-61 and 62-67, which are stretches known to be involved in the antiparallel beta-sheet at the center of the protein. Anal Chem, 1998 Aug 1, 70(15), 3235 - 41 Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization; Yang L et al.; On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins . The acquisition of high-resolution mass spectra of hemoglobin beta chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da . Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR . The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study. Cell Calcium, 2000 Jun, 27(6), 309 - 14 Delta 469 mutation in the type 3 repeat calcium binding domain of cartilage oligomeric matrix protein (COMP) disrupts calcium binding; Hou J et al.; Cartilage oligomeric matrix protein (COMP/TSP5), a large glycoprotein found in the territorial matrix surrounding chondrocytes, is the fifth member of the thrombospondin (TSP) gene family . While the function of COMP is unknown, its importance is underscored by the finding that mutations in the highly conserved type 3 repeat domain causes two skeletal dysplasias . Pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia, Fairbanks type (EDM1) . The type 3 repeats are highly conserved low-affinity Ca(2+)binding domains that are found in all TSP genes . This study was undertaken to determine the effects of mutations on calcium binding and structure of the type 3 repeat domains . Wild-type (WT) and Delta469 recombinant COMP (rCOMP) proteins containing the entire calcium-binding domain were expressed in E . coli and purified . Equilibrium dialysis demonstrated that WT bound 10-12 Ca(2+)ions/molecule while Delta469 bound approximately half the Ca(2+)ions . Circular dichroism (CD) spectrometry had striking spectral changes for the WT in response to increasing concentrations of Ca(2+) . These CD spectral changes were cooperative and reversible . In contrast, a large CD spectral change was not observed at any Ca(2+)concentration for Delta469 . Moreover, both WT and Delta469 proteins produced similar CD spectral changes when titrated with Zn(2+), Cu(2+)and Ni(2+)indicating that the Delta469 mutation specifically affects only calcium binding . These results suggest that the Delta469 mutation, in the type 3 repeat region, interferes with Ca(2+)binding and that filling of all Ca(2+)binding loops may be critical for correct COMP protein conformation . Hum Mutat . 2000 Oct;16(4):373. Identification and characterization of two novel mutations that produce acute intermittent porphyria: A 3-base deletion (841-843delGGA) and a missense mutation (T35M); De Siervi A et al.; A partial deficiency of Porphobilinogen deaminase (PBGD) is responsible for acute intermittent porphyria (AIP) . AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000 . Here, two new mutations and two previously reported were found in the PBGD gene in 22 Argentinean AIP patients corresponding to 8 different families . To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 6 PCR reactions, then all coding exons and flanking intronic regions were sequenced . The novel mutations are 841-843delGGA in exon 14, which results in the loss of glycine-281 (G281del), and one 104C>T point mutation in the exon 4 (T35M) . To further characterize both novel mutations, the pKK-PBGD construct for the mutant alleles were expressed in E . coli, the enzymatic activity of the recombinant proteins were 1% and 4% of the mean level expressed by the normal allele for 841-843delGGA and T35M, respectively . Hum Mutat 16:373, 2000 . Proteins, 2000, Suppl 4, 8 - 22 Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein; Kozlov AG et al.; Many macromolecular interactions, including protein-nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest . We have shown previously that temperature-dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, DeltaH(obs), and accounts for as much as a half of the observed heat capacity change, DeltaC(p) . However, there is still a substantial DeltaC(p) associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking . In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)(34) to SSB over a broad pH range (pH 5 . 0-10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25 degrees C), and as a function of temperature at pH 8.1 . A net protonation of the SSB protein occurs upon dT(pT)(34) binding over this entire pH range, with contributions from at least three sets of protonation sites (pK(a1) = 5.9-6.6, pK(a2) = 8.2-8.4, and pK(a3) = 10.2-10.3) and these protonation equilibria contribute substantially to the observed DeltaH and DeltaC(p) for the SSB-dT(pT)(34) interaction . The contribution of this coupled protonation ( approximately -260 to -320 cal mol(-1) K(-1)) accounts for as much as half of the total DeltaC(p) . The values of the "intrinsic" DeltaC(p,0) range from -210 +/- 33 cal mol(-1) degrees K(-1) to -237 +/- 36 cal mol(-1)K(-1), independent of {NaCl} . These results indicate that the coupling of a temperature-dependent protonation equilibria to a macromolecular interaction can result in a large negative DeltaC(p), and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand-macromolecular interactions, as well as protein folding. J Lipid Res, 2000 Oct, 41(10), 1629 - 39 24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A); Norlin M et al.; (24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ . Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol . In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver . 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol {(24S) and (24R)} was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes . In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol . 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A . Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer . Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols . Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids. J Biol Chem, 2000 Dec 22, 275(51), 40180 - 6 The thioredoxin system of the malaria parasite Plasmodium falciparum . Glutathione reduction revisited; Kanzok SM et al.; In most living cells, redox homeostasis is based both on the glutathione and the thioredoxin system . In the malaria parasite Plasmodium falciparum antioxidative proteins represent promising targets for the development of antiparasitic drugs . We cloned and expressed a thioredoxin of P . falciparum (pftrx), and we improved the stable expression of the thioredoxin reductase (PfTrxR) of the parasite by multiple silent mutagenesis . Both proteins were biochemically characterized and compared with the human host thioredoxin system . Intriguingly, the 13-kDa protein PfTrx is a better substrate for human TrxR (K(m) = 2 microm, k(cat) = 3300 min(-)(1)) than for P . falciparum TrxR (K(m) = 10.4 microm, k(cat) = 3100 min(-)(1)) . Possessing a midpoint potential of -270 mV, PfTrx was found to reduce the disease-related metabolites S-nitrosoglutathione and GSSG . The rate constant k(2) for the reaction between reduced P . falciparum thioredoxin and GSSG was determined to be 0.039 microm(-)(1) min(-)(1) at 25 degrees C and pH 7.4 . The k(2) for thioredoxins from man, Drosophila melanogaster, and Escherichia coli was approximately 5 times lower . Our data suggest that GSSG reduction can be supported at a high rate by the TrxR/Trx system in glutathione reductase-deficient cells; this may be relevant for certain stages of the malarial parasite but also for cells containing high {GSSG} of other organisms like dormant forms of Neurospora, glutathione reductase-deficient yeast mutants, or CD4(+) lymphocytes of AIDS patients. J Biol Chem, 2001 Jan 12, 276(2), 1369 - 75 The loop region covering the iron-sulfur cluster in bovine adrenodoxin comprises a new interaction site for redox partners; Hannemann F et al.; The amino acid in position 49 in bovine adrenodoxin is conserved among vertebrate {2Fe-2S} ferredoxins as hydroxyl function . A corresponding residue is missing in the cluster-coordinating loop of plant-type {2Fe-2S} ferredoxins . To probe the function of Thr-49 in a vertebrate ferredoxin, replacement mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49Delta, were generated and expressed in Escherichia coli . CD spectra of purified proteins indicate changes of the {2Fe-2S} center geometry only for mutant T49Delta, whereas NMR studies reveal no transduction of structural changes to the interaction domain . The redox potential of T49Delta (-370 mV) is lowered by approximately 100 mV compared with wild type adrenodoxin and reaches the potential range of plant-type ferredoxins (-305 to -455 mV) . Substitution mutants show moderate changes in the binding affinity to the redox partners . In contrast, the binding affinity of T49Delta to adrenodoxin reductase and cytochrome P-450 11A1 (CYP11A1) is dramatically reduced . These results led to the conclusion that Thr-49 modulates the redox potential in adrenodoxin and that the cluster-binding loop around Thr-49 represents a new interaction region with the redox partners adrenodoxin reductase and CYP11A1 . In addition, variations of the apparent rate constants of all mutants for CYP11A1 reduction indicate the participation of residue 49 in the electron transfer pathway between adrenodoxin and CYP11A1. EMBO J, 2000 Oct 2, 19(19), 5251 - 8 Subunit-specific degradation of the UmuD/D' heterodimer by the ClpXP protease: the role of trans recognition in UmuD' stability; Gonzalez M et al.; The Escherichia coli UmuD' protein is a subunit of the recently described error-prone DNA polymerase, pol V . UmuD' is initially synthesized as an unstable and mutagenically inactive pro-protein, UmuD . Upon processing, UmuD' assumes a relatively stable conformation and becomes mutagenically active . While UmuD and UmuD' by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers . Quite strikingly, it is in this context that UmuD' becomes susceptible to ClpXP-mediated proteolysis . Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD' subunit of the UmuD/D' heterodimer complex, while leaving the UmuD protein intact . Surprisingly, a signal that is essential and sufficient for targeting UmuD' for degradation was found to reside on UmuD not UmuD' . UmuD was also shown to be capable of channeling an excess of UmuD' to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error-prone DNA replication. EMBO J, 2000 Oct 2, 19(19), 5241 - 50 Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer; Diedrich G et al.; Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit . That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2 . L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center . However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs . Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions . None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation . These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis. EMBO J, 2000 Oct 2, 19(19), 5212 - 21 Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm; Qin Y et al.; Promoter binding by TraR and LuxR, the activators of two bacterial quorum-sensing systems, requires their cognate acyl-homoserine lactone (acyl-HSL) signals, but the role the signal plays in activating these transcription factors is not known . Soluble active TraR, when purified from cells grown with the acyl-HSL, contained bound signal and was solely in dimer form . However, genetic and cross-linking studies showed that TraR is almost exclusively in monomer form in cells grown without signal . Adding signal resulted in dimerization of the protein in a concentration-dependent manner . In the absence of signal, monomer TraR localized to the inner membrane while growth with the acyl-HSL resulted in the appearance of dimer TraR in the cytoplasmic compartment . Affinity chromatography indicated that the N-terminus of TraR from cells grown without signal is hidden . Analysis of heterodimers formed between TraR and its deletion mutants localized the dimerization domain to a region between residues 49 and 156 . We conclude that binding signal drives dimerization of TraR and its release from membranes into the cytoplasm. Kidney Int, 2000 Oct, 58(4), 1481 - 91 Mechanism of chronic obstructive uropathy: increased expression of apoptosis-promoting molecules; Choi YJ et al.; BACKGROUND: We have demonstrated that renal tubular and interstitial cells undergo pronounced apoptosis during the course of chronic obstructive uropathy (COU) . Apoptosis is a complex cellular process consisting of multiple steps, each of which is mediated by families of related molecules . These families may include receptor/ligand molecules such as Fas, Fas ligand, tumor necrosis factor receptor-1 (TNFR-1), and TNF-related apoptosis inducing ligand (TRAIL); signal transduction adapter molecules such as Fas-associated death domain (FADD), TNFR-1 associated death domain (TRADD), receptor-interacting protein (RIP), Fas-associated factor (FAF), and Fas-associated phosphatase (FAP); or effector molecules such as caspases . However, the mechanism of tubular cell apoptosis, as well as the pathogenetic relevance of these apoptosis-related molecules in COU, remains poorly understood . METHODS: Kidneys were harvested from sham-operated control mice and mice with COU created by left ureter ligation sacrificed in groups of three at days 4, 15, 30, and 45 . To detect apoptotic tubular and interstitial cells, in situ end labeling of fragmented DNA was performed . To detect the expression of apoptosis-related molecules, ribonuclease protection assay was used with specific antisense RNA probes for Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAF, FAP, and caspase-8 . Immunostaining for Fas, Fas ligand, TRAIL, TRADD, RIP, and caspase-8 was also performed . To assess the role of these molecules in COU-associated renal cell apoptosis, the frequencies of apoptotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated with those of apoptosis-related molecules . RESULTS: The obstructed kidneys displayed increased apoptosis of both tubular and interstitial cells . Tubular cell apoptosis appeared at day 4 after ureter ligation, peaked (fivefold of control) at day 15, and decreased gradually until the end of the experiment . In contrast, interstitial cell apoptosis sustained a progressive increase throughout the experiment . Apoptosis was minimal at all experimental time points for control and contralateral kidneys . Compared with control and contralateral kidneys, the ligated kidneys displayed a dynamic expression of mRNAs for many apoptosis-related molecules, which included an up to threefold increase for Fas, Fas ligand, TNF-R1, TRAIL, TRADD, RIP, and caspase-8, and an up to twofold increase for FADD and FAP, but there was little change for FAF . These mRNAs increased between days 4 and 15, decreased until day 30, but then increased again until day 45 . The rise and fall of mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis in that period . The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis . We demonstrated a positive immunostaining for Fas and Fas ligand in the tubular cells at early time points as well as in interstitial inflammatory cells at later time points . Although increased expression of TRAIL, TRADD, RIP, and caspase-8 was noted in tubular cells, there was no staining for these molecules in interstitial cells . CONCLUSION: The current study documents a dynamic expression of several molecules that are known to mediate the most crucial steps of apoptosis . It implicates these molecules in COU-associated renal cell apoptosis and in the pathogenesis of this condition . It also lays the foundation for interventional studies, including genetic engineering, to evaluate the molecular control of apoptosis associated with COU. Immunology, 2000 Oct, 101(2), 225 - 32 M-ficolin is expressed on monocytes and is a lectin binding to N-acetyl-D-glucosamine and mediates monocyte adhesion and phagocytosis of Escherichia coli; Teh C et al.; Ficolins are a group of multimeric proteins that contain collagen-like and fibrinogen-like (FBG) sequences . Three types of ficolins have been characterized: H-, L- and M-ficolins . Both H- and L-ficolins have demonstrated lectin activities . In the present study, the FBG domain of M-ficolin was expressed and shown to bind to N-acetyl-D-glucosamine . M-ficolin mRNA was expressed in monocytes but not in the more differentiated macrophages and dendritic cells . By flow cytometry, surface biotinylation and immunoprecipitation, we showed that M-ficolin was associated with the surface of promonocytic U937 cells . M-ficolin transiently expressed in COS-7 cells was also clearly detected on the cell surface by immunoprecipitation . By flow cytometry, M-ficolin was detected on peripheral blood monocytes but not on lymphocytes or granulocytes . Immobilized rabbit anti-M-ficolin F(ab')2 mediated U937 cell adhesion, and the antibody also inhibited phagocytosis of Escherichia coli K-12 by U937 cells . Therefore, M-ficolin might act as a phagocytic receptor or adaptor on circulating monocytes for micro-organism recognition and may potentially mediate monocyte adhesion. Immunology, 2000 Sep, 101(1), 154 - 60 Genetically detoxified mutants of heat-labile enterotoxin from Escherichia coli are effective adjuvants for induction of cytotoxic T-cell responses against HIV-1 gag-p55; Neidleman JA et al.; There is an urgent need for prophylactic and therapeutic vaccines against human immunodeficiency virus (HIV) . Mucosal immunization strategies have great potential to elicit both mucosal and systemic cellular immunity required to protect against HIV-induced acquired immune deficiency syndrome (AIDS) . However, mucosal immunizations with soluble protein antigens generally require adjuvants . In this study, we tested two mutants of the heat-labile enterotoxin (LT) from Escherichia coli, LTK63: with no measurable ADP-ribosyltransferase activity, and LTR72: with residual ADP-ribosyltransferase activity, as mucosal adjuvants for induction of cytotoxic T lymphocyte (CTL) responses to coadministered HIV gag p55 protein . We found that intranasal (i.n.) immunizations with HIV gag p55 protein coadministered with LTK63 or LTR72 induced systemic CTL responses comparable to that obtained following intramuscular (i . m.) immunizations with the same adjuvants . Moreover, oral coadministration of LTR72, but not LTK63, resulted in local as well as systemic p55-specific CTL responses in mesenteric lymph nodes (MLN) and spleens (SP) of the immunized mice . These data have important implications for current efforts to develop a safe vaccine against HIV. Eur J Biochem, 2000 Oct, 267(20), 6311 - 8 Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides; Guay J et al.; The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes . An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation . The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli . The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage . All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain . Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism . The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S . The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM) . Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM) . This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L {Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J . E., Weber, E . & Wiederanders, B . (1997), Eur J . Biochem . 250, 745-750} . These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs . the parent enzymes, but selective inhibition vs . cathepsin B and papain was obtained. Eur J Biochem, 2000 Oct, 267(20), 6302 - 10 Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids . Effects on catalytic and stability properties; Solstad T et al.; The microheterogeneity of recombinant human phenylalanine hydroxylase (hPAH) was investigated by isoelectric focusing and 2D electrophoresis . When expressed in Escherichia coli four main components (denoted hPAH I-IV) of approximately 50 kDa were observed on long-term induction at 28-37 degrees C with isopropyl thio-beta-D-galactoside (IPTG), differing in pI by about 0.1 pH unit . A similar type of microheterogeneity was observed when the enzyme was expressed (1 h at 37 degrees C) in an in vitro transcription-translation system, including both its nonphosphorylated and phosphorylated forms which were separated on the basis of a difference in mobility on SDS/PAGE . Experimental evidence is presented that the microheterogeneity is the result of nonenzymatic deamidations of labile amide containing amino acids . When expressed in E . coli at 28 degrees C, the percentage of the acidic forms of the enzyme subunit increased as a function of the induction time with IPTG, representing about 50% on 8 h induction . When the enzyme obtained after 2 h induction (containing mainly hPAH I) was incubated in vitro, its conversion to the acidic components (hPAH II-IV) revealed a pH and temperature dependence characteristic of a nonenzymatic deamidation of asparagine residues in proteins, with the release of ammonia . Comparing the microheterogeneity of the wild-type and a truncated form of the enzyme expressed in E . coli, it is concluded that the labile amide groups are located in the catalytic domain as defined by crystal structure analysis {Erlandsen, H., Fusetti, F., Martinez, A., Hough, E., Flatmark, T . & Stevens, R . C . (1997) Nat . Struct . Biol . 4, 995-1000} . It is further demonstrated that the progressive deamidations which occur in E . coli results in a threefold increase in the catalytic efficiency (Vmax/{S}0.5) of the enzyme and an increased susceptibility to limited tryptic proteolysis, characteristic of a partly activated enzyme . The results also suggest that deamidation may play a role in the long term regulation of the catalytic activity and the cellular turnover of this enzyme. Eur J Biochem, 2000 Oct, 267(20), 6239 - 48 Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones; Rial DV et al.; We have analyzed the interaction of DnaK and plant Hsp70 proteins with the wild-type ferredoxin-NADP+ reductase precursor (preFNR) and mutants containing amino-acid replacements in the targeting sequence . Using an algorithm already developed {Rudiger, S., Germeroth, L., Schneider-Mergener, J . & Bukau, B . (1997) EMBO J . 16, 1501-1507} we observed that 75% of the 727 plastid precursor proteins analyzed contained at least one site with high likelihood of DnaK binding in their transit peptides . Statistical analysis showed a decrease of DnaK binding site frequency within the first 15 amino-acid residues of the transit peptides . Using fusion proteins we detected the interaction of DnaK with the transit peptide of the folded preFNR but not with the mature region of the protein . Discharge of DnaK from the presequence was favored by addition of MgATP . When a putative DnaK binding site was artificially added at the N-terminus of the mature protein, we observed formation of complexes with bacterial and plant Hsp70 molecular chaperones . Reducing the likelihood of DnaK binding by directed mutagenesis of the presequence increased the release of bound DnaK . The Hsp70 proteins from plastids and plant cell cytosol also interacted with the preFNR transit peptide . Overall results are discussed in the context of the proposed models to explain the organelle protein import. Eur J Biochem, 2000 Oct, 267(20), 6188 - 96 A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae; Kwon TH et al.; Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus {Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S . , Kawabata, S.I., Iwanaga, S . & Lee, B.L . (1998) Eur . J . Biochem . 257, 615-621} . PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development . Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae . The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da . The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster . The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus . A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor . By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system . However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed . Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-kDa protein was cleaved to a 35-kDa protein . RNA blot hybridization revealed that expression of the 45-kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge. Eur J Biochem, 2000 Oct, 267(20), 6158 - 65 Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24; Sakaki T et al.; Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed . The reconstituted system containing the membrane fraction prepared from recombinant E . coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds . Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway {Sakaki, T . Sawada, N . Nonaka, Y . Ohyama, Y . & Inouye, K . (1999) Eur . J . Biochem . 262, 43-48} . HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3 . On 1alpha,25(OH)2D3 metabolism, similar results were observed . These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3 . We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E . coli . Addition of 25(OH)D3 to the recombinant E . coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways . Thus, the E . coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. Eur J Biochem, 2000 Oct, 267(20), 6118 - 25 Thioredoxin reductase as a pathophysiological factor and drug target; Becker K et al.; Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents . As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2-based oxidative stress . Being a selenite-reducing enzyme as well as a selenol-containing enzyme, human TrxR plays a central role in selenium (patho)physiology . Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR . Selenocysteine 496 of human TrxR is a major target of the anti-rheumatic gold-containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nM . The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjogren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence . Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2 . The effects of anti-tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR . Consistently, high levels of the enzyme can support drug resistance . TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction . This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria. J Crit Care, 2000 Sep, 15(3), 97 - 102 Influence of amrinone on intestinal villus blood flow during endotoxemia; Schmidt W et al.; PURPOSE: The objective of this study was to determine the effects of a continuous infusion of the phosphodiesterase (PDE) inhibitor amrinone on mucosal villus blood flow in a normotensive model of endotoxemia . MATERIALS AND METHODS: Twenty-four anesthetized and ventilated rats underwent laparotomy, and an ileal portion was exteriorized and opened by an antimesenteric incision . The ileal segment was fixed on a plexiglass stage with the mucosal surface upward . Microcirculatory parameters were assessed by intravital videomicroscopy . The animals were randomly assigned to receive one of three treatments: infusion of Escherichia coli lipopolysaccharides (LPS, 2 mg/kg/h) without phosphodiesterase inhibitor pretreatment (LPS group); or infusion of LPS with amrinone pretreatment (40 microg/kg/min, start 30 minutes before LPS infusion) (amrinone group), or infusion of equivalent volumes of NaCl 0.9% (control group) . Macrohemodynamic parameters (MAP, HR) and microhemodynamic parameters of ileal mucosa (mean diameter of central arterioles = D(A) and mean erythrocyte velocity within the arterioles = VE) were measured 30 minutes before and at 0, 60, and 120 minutes after induction of endotoxemia . Mucosal villus blood flow was calculated from D(A) and VE . RESULTS: In this normotensive endotoxemia model, MAP remained stable in the control and the LPS group but significantly decreased in the amrinone group.The endotoxin-induced decrease of V(E) and D(A) of central arterioles of mucosal villi could be attenuated and prevented, respectively . Thus, the endotoxin-induced decrease of mucosal villus blood flow was diminished but not fully restored by amrinone infusion . CONCLUSION: Our results indicate that amrinone during an early stage of sepsis is of limited value . It attenuates mucosal hypoperfusion but contributes to systemic hypotension. Microb Comp Genomics, 2000, 5(1), 7 - 15 Relationship between whole proteome aminoacid composition and static DNA curvature; Jauregui R et al.; To study possible relationships between an organism's genomic DNA curvature and the aminoacid composition of its proteome, every peptidic sequence from fully determined genomes was retrotranslated using the E . coli codon preferences, and the curvature profiles of the resulting DNA sequences were calculated and compared . A clear interdependence between these two variables was observed, as each retrotranslated proteome presented a distinctive, statistically significant DNA curvature profile biased toward its natural DNA curvature profile . In addition, by comparing the profiles arising from real and randomly permuted proteomes, we also found a position-dependent contribution of the peptidic sequence to DNA curvature . The implications of these results support the idea of a possible selection toward a specific global curvature of genomes.
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