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 considera