Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2373 - 80 Gene cloning and function analysis of replication factor C from Thermococcus kodakaraensis KOD1; Kitabayashi M et al.; Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase . The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells . T . kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa) . The DNA elongation rate of the family B DNA polymerase from T . kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T . kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA. Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2359 - 64 A thermostable non-xylanolytic alpha-glucuronidase of Thermotoga maritima MSB8; Suresh C et al.; A putative alpha-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase, not alpha-glucosidase . It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides . Agu4B was thermostable with an optimum temperature of 80 degrees C . It strictly required Mn(2+) and thiol compounds for its activity . The presence of NAD(+) slightly activated the enzyme . The amino acid sequence of Agu4B showed higher identity with Agu4A (another alpha-glucuronidase of T . maritima, 61%) than with AglA (alpha-glucosidase of T . maritima, 48%). J Synchrotron Radiat, 2004 Jan 1, 11(Pt 1), 89 - 92 Epub 2003 Nov 28. Virus structure analysis with synchrotron radiation: methods and results; Johnson JE; Structural studies of viruses that are investigated as part of a program to understand molecular machines are described . Crystallography, solution X-ray scattering, electron microscopy and molecular virology were employed to investigate structure, assembly and maturation of RNA and dsDNA viruses . 240 copies of the RNA viral subunits (each with 650 amino acids) spontaneously assemble at pH 7 in a baculovirus expression system to form T = 4 icosahedral particles, 450 A in diameter . At pH 5 the particles condense to 410 A and the subunits auto-catalytically cleave at residue 570 . 420 copies of the dsDNA viral subunits (281 amino acids each) assemble in an E . coli expression system to form T = 7 icosahedral particles, 450 A in diameter . At pH 4 the particles expand to 650 A diameter with the auto-catalytic formation of cross links between a lysine side chain and an asparagine side chain creating a concatenated set of 60 hexamer and 12 pentamer rings rendering the particle impervious to denaturation without protease treatment . Moderate- to high-resolution structures of the procapsids and capsids of these virus particles have been determined as well as cryoEM reconstructions of intermediate structures in order to define the driving force and the trajectories of these large-scale transitions. J Synchrotron Radiat, 2004 Jan 1, 11(Pt 1), 86 - 8 Epub 2003 Nov 28. Water and glycerol permeation through the glycerol channel GlpF and the aquaporin family; Lee JK et al.; The 2.2 A resolution crystal structure of GlpF, an E . coli aquaporin that facilitates the flow of glycerol, water and other small solutes, provides much insight into the molecular function and selectivity of aquaporins . Using GlpF and its atomic structure as a paradigm for the ten highly conserved human aquaporins, site-directed mutagenesis has been used to mutate residues that are possibly integral to the structure and function of different aquaporins . X-ray crystallography and other biophysical and molecular simulation methods allows for assessment of these changes at the structural and functional level . Initial attempts to convert the glycerol specific properties of GlpF towards a water specific aquaporin resulted in the shifting of GlpF channel properties towards that of the water aquaporins . This result reveals the great possibility of emulating and deciphering the function of other aquaporins with GlpF via mutagenesis and investigation of structure and function. J Synchrotron Radiat, 2004 Jan 1, 11(Pt 1), 1 - 3 Epub 2003 Nov 28. Overview and new developments in softer X-ray (2A < lambda < 5A) protein crystallography; Helliwell JR; New methodologies with synchrotron radiation and X-ray free electron lasers (XFELs) in structural biology are being developed . Recent trends in harnessing softer X-rays in protein crystallography for phase determination are described . These include reference to a data-collection test at 2.6 A wavelength with a lysozyme crystal on SRS station 7.2 (Helliwell, 1983) and also use of softer X-rays (2 A wavelength) to optimise f " at the xenon L1 absorption edge in the Single Isomorphous Replacement Optimised Anomalous Scattering ('SIROAS') structure determination of apocrustacyanin A1 with four, partially occupied, xenon atoms (Cianci et al., 2001; Chayen et al., 2000) . The hand of the protein was determined using the f " enhanced sulphur anomalous signal from six disulphides in the protein dimer of 40 kDa . In a follow-up study the single wavelength xenon L1-edge f " optimised data set alone was used for phase determination and phase improvement by solvent flattening etc . (CCP4 DM) (Olczak et al., 2003) . Auto-tracing of the protein was feasible but required additional diffraction data at higher resolution . This latter could be avoided in future by using improved tilted detector settings during use of softer X-rays, i.e . towards back-scattering recording (Helliwell, 2002) . The Olczak et al . study has already led to optimisation of the new SRS beamline MPW MAD 10 (see firstly involving the thinning of the beryllium windows as much as possible and planning for a MAR Research tilted detector 'desk top beamline' geometry . Thus the use of softer, i.e . 2 to 3 A wavelength range, X-rays will allow optimisation of xenon and iodine L-edge f " and enhancing of sulphur f " signals for higher throughput protein crystallography . Softer X-rays utilisation in protein crystallography includes work done on SRS bending-magnet station 7.2 in the early 1980s by the author as station scientist (Helliwell, 1984) . In the future development of XFELs these softer X-ray wavelengths could also be harnessed and relax the demands to some extent on the complexity and cost of an XFEL . Thus, by use of say 4 A XFEL radiation and use of a back-scattering geometry area detector the single molecule molecular transform could be sampled to a spatial resolution of 2 A, sufficient, in principle, for protein model refinement (Miao et al., 1999) . Meanwhile, Miao et al . (2003) report the first experimental recording of the diffraction pattern from intact Escherichia coli bacteria using coherent X-rays, with a wavelength of 2 A, at a resolution of 30 nm and a real-space image constructed . The new single-particle X-ray diffraction-imaging era has commenced. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2348 - 52 Epub 2003 Nov 27. Molecules of Escherichia coli MobB assemble into densely packed hollow cylinders in a crystal lattice with 75% solvent content; Rangarajan SE et al.; The crystal structure of Escherichia coli MobB, an enzyme involved in the final step of molybdenum-cofactor biosynthesis, forms intertwined dimers . Each molecule consists of two segments and requires the second monomer for stable folding . Dimerization buries a quarter of the solvent-accessible area of the monomer . These dimers assemble into a hexagonal lattice with P6(4)22 symmetry and occupy only approximately 25% of the unit-cell volume . The symmetry-related dimers associate tightly into a helical structure with a diameter of 250 A and a pitch of 98 A . Two such helices are intertwined, shifted by 49 A along the sixfold axis . Within the crystal, these helices form thin-walled cylinders with an external diameter of 250 A and an internal diameter of 190 A . Their center is filled with solvent . These cylinders pack closely together, forming a hexagonal lattice with the highest possible packing density . This arrangement of dimers allows extensive intermolecular contacts with 75% solvent content in the crystal. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2332 - 3 Epub 2003 Nov 27. Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase V; Arita K et al.; Human peptidylarginine deiminase V (PAD V) is a post-translational enzyme that catalyzes the conversion of arginine residues in protein into citrulline residues in the presence of calcium ion . Crystals of PAD V have been grown at 293 K using polyethylene glycol monomethylether as a precipitant . Crystals diffracted beyond 2.7 A resolution at 100 K at the SPring-8 synchrotron-radiation source . The crystal belongs to space group C2, with unit-cell parameters a = 144.6, b = 60.4, c = 113.4 A, beta = 123.6 degrees . The asymmetric unit contains one molecule, with a V(M) of 2.56 A(3) Da(-1) and a solvent content of 56.1% . A full set of X-ray diffraction data was collected to 2.8 A resolution with a completeness of 97.5% . Heavy-atom derivatives have been successfully prepared and structure analysis is in progress. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2325 - 7 Epub 2003 Nov 27. Crystallization of {Fe3S4}-ferredoxin from the hyperthermophile archaeon Pyrococcus furiosus; Nielsen MS et al.; Recombinant Pyrococcus furiosus ferredoxin with a {Fe3S4}-cluster was crystallized through steps of optimization and X-ray diffraction data were collected from several crystal forms . Flat plate-like crystals were grown by hanging-drop vapour diffusion . The precipitant used was 30% PEG 400; the pH was varied between 7.0 and 8.0 and hexaamminecobalt(III) chloride was essential for crystal formation . The best crystals belong to space group P2(1), with unit-cell parameters a = 31.23, b = 47.51, c = 52.03 A, beta = 103.86 degrees, and diffract to 1.5 A resolution. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2316 - 8 Epub 2003 Nov 27. Crystallization and preliminary X-ray analysis of N-acetyl-1-D-myo-inosityl-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis; McCarthy AA et al.; Mycobacteria synthesize mycothiol (MSH) as a low-molecular-weight thiol that protects against oxidative stress in a similar role to that of glutathione in many other species . The absence of MSH in mammals suggests that enzymes from its biosynthetic pathway in Mycobacterium tuberculosis could be useful targets for drug design . The gene for MshB (Rv1170), the enzyme that catalyses the second step in MSH biosynthesis in M . tuberculosis, has been cloned and the protein has been expressed in Escherichia coli both in native and SeMet-substituted forms and crystallized in two crystal forms . One of these, prepared in the presence of beta-octylglucoside as a key additive, is suitable for high-resolution X-ray structural analysis . The crystals are orthorhombic, with unit-cell parameters a = 71.69, b = 83.74, c = 95.65 A, space group P2(1)2(1)2(1) and two molecules in the asymmetric unit . X-ray diffraction data to 1.9 A resolution have been collected. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2313 - 5 Epub 2003 Nov 27. Crystallization of CprB, an autoregulator-receptor protein from Streptomyces coelicolor A3(2); Natsume R et al.; CprB, an autoregulator-receptor protein from Streptomyces coelicolor A3(2), was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 6000 as a precipitant . Three crystal forms (I, II and III) were obtained; crystal forms I and II were useful for structure determination . Form I crystals belong to an orthorhombic system with space group P2(1)2(1)2(1) and diffracted to better than 2.4 A . Form II crystals belong to a tetragonal system with space group P4(1(3))2(1)2. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2303 - 5 Epub 2003 Nov 27. Crystallization and preliminary X-ray diffraction data of Mycobacterium tuberculosis FbpC1 (Rv3803c); Wilson RA et al.; The heterotrimeric antigen 85 complex (Ag85) is a major component of the cell wall of Mycobacterium tuberculosis and consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2) . These play key roles in the pathogenesis of tuberculosis and in maintaining cell-wall integrity . A homologue of the Ag85 subunits ( approximately 40% identity) was recently annotated in the M . tuberculosis genome as FbpC1 . Unlike the Ag85-complex components, FbpC1 lacks mycolyltransferase activity and its function remains to be established . In order to aid functional characterization, FbpC1 has been crystallized . At room temperature, tetragonal crystals of FbpC1 were obtained belonging to space group P4(1)2(1)2 (unit-cell parameters a = b = 109.9, c = 61.8 A), yet when frozen the crystals underwent a phase transition to orthorhombic symmetry, space group P2(1)2(1)2(1) (a = 59.9, b = 108.9, c = 109.9 A) . Diffraction data complete to 1.7 A resolution were recorded at 100 K at the synchrotron. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2297 - 9 Epub 2003 Nov 27. Crystallization and preliminary crystallographic analysis of human serine dehydratase; Sun L et al.; L-Serine dehydratase (SDH) catalyzes the pyridoxal phosphate (PLP) dependent deamination of L-serine to yield pyruvate . Recombinant human serine dehydratase was crystallized by the hanging-drop vapour-diffusion method . Crystals were grown at 291 K using (NH4)(2)SO4 as precipitant . Diffraction data were obtained to a resolution of 2.5 A from a single frozen crystal using Cu Kalpha radiation . The crystal belongs to space group I422, with unit-cell parameters a = 157.4, b = 157.4, c = 59.2 A, alpha = beta = gamma = 90 degrees . The asymmetric unit contains one molecule and has a solvent content of about 46%. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2286 - 8 Epub 2003 Nov 27. Expression, crystallization and preliminary X-ray crystallographic studies of Arthrobacter globiformis inulin fructotransferase; Momma M et al.; A recombinant form of Arthrobacter globiformis inulin fructotransferase (DFAIII-producing) has been overexpressed in Escherichia coli and purified to homogeneity . Crystals were obtained at 293 K by the hanging-drop vapour-diffusion technique using 0.1 M Na HEPES pH 7.5 buffer containing 1.5 M lithium sulfate as a precipitant . Crystals of the recombinant wild-type enzyme diffracted to better than 1.5 A at 100 K using a synchrotron-radiation source at the Photon Factory . The crystal belonged to space group R32, with unit-cell parameters a = b = 92.02, c = 229.82 A in the hexagonal axes . Assuming the presence of one molecule in the asymmetric unit, the V(M) value for the crystal was 2.15 A(3) Da(-1), indicating a solvent content of 42.8% . Selenomethionine-derivative crystals belonged to a different space group, C2, with unit-cell parameters a = 159.32, b = 91.92, c = 92.58 A, beta = 125.06 . Matthews coefficient calculations suggested that the C2 selenomethionine-derivative crystal contained three molecules per asymmetric unit. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2272 - 4 Epub 2003 Nov 27. Purification, crystallization and preliminary X-ray diffraction analysis of S-formylglutathione hydrolase from Arabidopsis thaliana: effects of pressure and selenomethionine substitution on space-group changes; McAuley KE et al.; S-Formylglutathione hydrolase (SFGH) has activity toward several xenobiotic carboxyesters and catalyses the final step of formaldehyde detoxification: the hydrolysis of S-formylglutathione to formate and glutathione . The Arabidopsis thaliana enzyme (AtSFGH) was crystallized in space group C2, with unit-cell parameters a = 128.5, b = 81.1, c = 94.3 A, beta = 93.3 degrees and three molecules in the asymmetric unit . A second crystal form of AtSFGH could be obtained by pressurizing the monoclinic crystals at 2 MPa for 30 min . The resulting space group is either P3(1)21 or P3(2)21, with unit-cell parameters a = 75.1, c = 92.8 A and one molecule in the asymmetric unit . Crystallographic data have been collected for both crystal forms to resolutions of 1.7 A for the monoclinic crystal and 1.6 A for the trigonal crystal . The structure has been solved by MAD phasing using a three-wavelength data set collected from a monoclinic crystal of selenomethionine-labelled AtSFGH. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2269 - 71 Epub 2003 Nov 27. Crystallization and preliminary crystallographic studies of the C-terminal domain of human FKBP52; Wu B et al.; FKBP52 is a high-molecular-weight immunophilin belonging to the FKBP (FK506-binding protein) family . FKBP52 is one of several chaperone proteins associated with untransformed steroid receptors in steroid receptor-hsp90 heterocomplexes . Here, the C-terminal domain (amino acids 145-459) has been cloned, overexpressed and purified . Crystals were obtained using the hanging-drop vapour-diffusion technique with ammonium sulfate as precipitant in 0.1 M Tris pH 8.0 solution . Diffraction data to 2.7 A were collected from a selenomethionine-containing crystal belonging to space group C222(1), with unit-cell parameters a = 114.4, b = 143.1, c = 171.2 A, alpha = beta = gamma = 90 degrees . There are three molecules per asymmetric unit. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2262 - 4 Epub 2003 Nov 27. Crystallization of RusA Holliday junction resolvase from Escherichia coli; Muranova TA et al.; Crystals of the Escherichia coli Holliday junction resolvase RusA have been obtained using the hanging-drop method and characterized . The crystals have a primitive monoclinic form and belong to space group P2(1) . The V(M) value suggests the presence of two copies of the monomer in the asymmetric unit . A full three-wavelength MAD data collection on a selenomethionine-incorporated form has been undertaken and structure determination is under way using data collected to 2.1 A resolution. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2259 - 61 Epub 2003 Nov 27. Expression and crystallographic characterization of the extracellular domain of human natural killer cell triggering receptor NKp46; Ponassi M et al.; Human natural killer (NK) cells are regulated in their cytolytic activity by a delicate interplay between activating and inhibitory signals related to distinct families of triggering and inhibitory receptor proteins . NKp46 is a major NK cell-specific triggering receptor involved in the recognition and lysis of human and murine tumour and virally infected cells . It consists of an extracellular portion, composed of two Ig-like domains, a transmembrane segment and a small cytoplasmic domain . To shed light on the molecular-recognition events involved in NK cytotoxicity triggering mechanisms, the NKp46 extracellular region was cloned, overexpressed, refolded and crystallized . X-ray diffraction data could be collected to a resolution limit of 1.93 A . Crystals of the NKp46 extracellular region belong to the hexagonal space group P6(1) (or P6(5)), with unit-cell parameters a = b = 85.48, c = 59.91 A, gamma = 120 degrees; the asymmetric unit contains one protein chain (197 amino acids). Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2254 - 6 Epub 2003 Nov 27. Crystallization and preliminary X-ray diffraction analysis of Mycobacterium smegmatis Dps; Roy S et al.; Three crystal forms of a DNA-binding protein from stationary phase cells (Dps) of Mycobacterium smegmatis have been grown . They are hexagonal, tetragonal and cubic . The structure of the cubic form, which has one subunit in the asymmetric unit, has been solved using the molecular-replacement method . The application of the crystallographic symmetry operations on the subunit leads to eight dodecamers with 23 symmetry in the unit cell. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2247 - 50 Epub 2003 Nov 27. Purification, crystallization and X-ray diffraction analysis of the extracellular part of the human Fc receptor for IgA, FcalphaRI (CD89); Wenig K et al.; FcalphaRI is the predominant receptor for IgA in the serum . Nevertheless, the interaction between the molecules that finally leads to an immune response is poorly understood . To investigate the structural requirements for IgA binding, the extracellular region of FcalphaRI was cloned and overexpressed in Escherichia coli . The resulting inclusion-body protein was refolded and purified . Despite its deglycosylated state, this recombinant FcalphaRI retained its ability to bind human IgA . The protein crystallized spontaneously as microcrystalline needles . Recrystallization yielded crystals belonging to a primitive monoclinic space group . A complete 2.8 A resolution X-ray diffraction data set was collected using synchrotron radiation. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2242 - 6 Epub 2003 Nov 27. Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasing; Lott JS et al.; The protein PAE2307 is a member of a protein family of unknown function which is conserved among a number of bacterial and archaeal species . The protein was overexpressed in Escherichia coli, purified and crystallized in two crystal forms . The prevalent form was twinned, but the other diffracted to 1.45 A resolution . The non-twinned crystals proved difficult to reproduce, so screening of potential heavy-atom derivatives by native polyacrylamide gel electrophoresis was used to establish suitable derivatization conditions . This process enabled the production of a K(2)Pt(NO2)4 derivative that was used to collect a single-wavelength anomalous diffraction (SAD) data set from the only available crystal . Phase information of high quality was obtained, enabling the calculation of an interpretable electron-density map. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2191 - 9 Epub 2003 Nov 27. Structure of superoxide dismutase from Pyrobaculum aerophilum presents a challenging case in molecular replacement with multiple molecules, pseudo-symmetry and twinning; Lee S et al.; The crystal structure of superoxide dismutase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was determined by molecular replacement at 1.8 A resolution . The structure determination was made especially challenging by the large number of molecules (24) in the asymmetric unit, the presence of a pseudo-crystallographic twofold operator close to a twinning operator and the inability to detect twinning by conventional means . Molecular replacement proceeded at low resolution in pseudo (apparent) space group P3(2)12 and was facilitated by examination of the self-rotation function and native Patterson map . Refinement, however, stalled at an R factor of 40% when high-resolution data were included . Expanding to the lower symmetry space group P3(2) decreased R (to 22%) and R(free) (to 26%), but not by as much as expected for the quality of data . Finally, despite the apparent lack of evidence from conventional twinning tests {i.e . plots of the second moment of I and N(Z) distributions}, a twinning operator was included in the refinement, lowering R and R(free) to 16.2 and 21.7%, respectively . The early detection of twinning appears to have been masked by a deviation in the expected intensity distribution caused by the presence of non-crystallographic translational symmetry . These findings suggest the importance of testing twinning operators in cases where pseudo-translational symmetry can explain negative results from conventional twinning tests . The structure reveals a tetrameric assembly with 222 symmetry, similar to superoxide dismutase structures from other organisms . The current structural model represents the metal-free state of the enzyme. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2183 - 90 Epub 2003 Nov 27. The structures of the PII proteins from the cyanobacteria Synechococcus sp . PCC 7942 and Synechocystis sp . PCC 6803; Xu Y et al.; The PII proteins from the cyanobacteria Synechococcus sp . PCC 7942 and Synechocystis sp . PCC 6803 have been crystallized and high-resolution structures have been obtained using X-ray crystallography . The core of these new structures is similar to that of the PII proteins from Escherichia coli, although the structures of the T- and C-loops differ . The T-loop of the Synechococcus protein is ordered, but appears to be stabilized by crystal contacts . The same loop in the Synechocystis protein is disordered . The C-terminus of the Synechocystis protein is stabilized by hydrogen bonding to the same region of a crystallographically related molecule . The same terminus in the Synechococcus protein is stabilized by coordination with a metal ion . These observations are consistent with the idea that both the T-loop and the C-terminus of PII proteins are flexible in solution and that this flexibility may be important for receptor recognition . Sequence comparisons are used to identify regions of the sequence unique to the cyanobacteria. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2133 - 9 Epub 2003 Nov 27. Three-dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2; Pechkova E et al.; The three-dimensional crystal structure of the triple-point mutant of the catalytic subunit of human protein kinase CK2alpha has been determined at 2.4 A resolution . Microcrystals of mutant CK2 catalytic subunit were obtained by a protein-crystallization method based on thin-film nanotechnology . These microcrystals (of about 20 micro m in diameter) were used for diffraction data collection by means of the microfocus beamline at the ESRF synchrotron . A comparison between the human protein kinase CK2alpha and the corresponding enzyme from a lower organism (Zea mays) is made. Science, 2003 Nov 28, 302(5650), 1558 - 60 High deleterious genomic mutation rate in stationary phase of Escherichia coli; Loewe L et al.; In natural habitats, bacteria spend most of their time in some form of growth arrest . Little is known about deleterious mutations in such stages, and consequently there is limited understanding of what evolutionary events occur . In a deleterious mutation accumulation experiment in prolonged stationary phase of Escherichia coli, about 0.03 slightly deleterious mutations were observed per genome per day . This is over an order of magnitude higher than extrapolations from fast-growing cells, but in line with inferences from observations in adaptive stationary phase mutation experiments . These findings may affect understanding of bacterial evolution and the emergence of bacterial pathogenicity. J Bacteriol, 2003 Dec, 185(24), 7085 - 91 Suppression of factor-dependent transcription termination by antiterminator RNA; King RA et al.; Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites . We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein . Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put . put-mediated antitermination thus shows no apparent terminator specificity, suggesting that put inhibits a step that is common to termination at the different types of terminator. Biophys J, 2003 Dec, 85(6), 4110 - 21 Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging; Morrison IE et al.; Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles . The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function . We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules . This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging . The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules . Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules . A method of correcting for positional shifts that result from chromatic aberration is presented . The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters . We have obtained preliminary data for colocalization of molecules on intact cells . Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described. Biophys J, 2003 Dec, 85(6), 3802 - 12 Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry; Hanson CL et al.; The interactions between phospholipid molecules in suspensions have been studied by using mass spectrometry . Electrospray mass spectra of homogeneous preparations formed from three different phospholipid molecules demonstrate that under certain conditions interactions between 90 and 100 lipid molecules can be preserved . In the presence of apolipoprotein C-II, a phospholipid binding protein, a series of lipid molecules and the protein were observed in complexes . The specificity of binding was demonstrated by proteolysis; the resulting mass spectra reveal lipid-bound peptides that encompass the proposed lipid-binding domain . The mass spectra of heterogeneous suspensions and their complexes with apolipoprotein C-II demonstrate that the protein binds simultaneously to two different phospholipids . Moreover, when apolipoprotein C-II is added to lipid suspensions formed with local concentrations of the same lipid molecule, the protein is capable of remodeling the distribution to form one that is closer to a statistical arrangement . These observations demonstrate a capacity for apolipoprotein C-II to change the topology of the phospholipid surface . More generally, these results highlight the fact that mass spectrometry can be used to probe lipid interactions in both homogeneous and heterogeneous suspensions and demonstrate reorganization of the distribution of lipids upon surface binding of apolipoprotein C-II. Biophys J, 2003 Dec, 85(6), 3718 - 29 Residue ionization and ion transport through OmpF channels; Nestorovich EM et al.; Single trimeric channels of the general bacterial porin, OmpF, were reconstituted into planar lipid membranes and their conductance, selectivity, and open-channel noise were studied over a wide range of proton concentrations . From pH 1 to pH 12, channel transport properties displayed three characteristic regimes . First, in acidic solutions, channel conductance is a strong function of pH; it increases by approximately threefold as the proton concentration decreases from pH 1 to pH 5 . This rise in conductance is accompanied by a sharp increase in cation transport number and by pronounced open-channel low-frequency current noise with a peak at approximately pH 2.5 . Random stepwise transients with amplitudes at approximately 1/5 of the monomer conductance are major contributors to this noise . Second, over the middle range (pH 5/pH 9), channel conductance and selectivity stay virtually constant; open channel noise is at its minimum . Third, over the basic range (pH 9/pH 12), channel conductance and cation selectivity start to grow again with an onset of a higher frequency open-channel noise . We attribute these effects to the reversible protonation of channel residues whose pH-dependent charge influences transport by direct interactions with ions passing through the channel. Brain Res, 2004 Jan 2, 995(1), 84 - 96 A survival motor neuron:tetanus toxin fragment C fusion protein for the targeted delivery of SMN protein to neurons; Francis JW et al.; Spinal muscular atrophy (SMA) is a degenerative disorder of spinal motor neurons caused by homozygous mutations in the survival motor neuron (SMN1) gene . Because increased tissue levels of human SMN protein (hSMN) in transgenic mice reduce the motor neuron loss caused by murine SMN knockout, we engineered a recombinant SMN fusion protein to deliver exogenous hSMN to the cytosolic compartment of motor neurons . The fusion protein, SDT, is comprised of hSMN linked to the catalytic and transmembrane domains of diphtheria toxin (DTx) followed by fragment C of tetanus toxin (TTC) . Following overexpression in Escherichia coli, SDT possessed a subunit molecular weight of approximately 130 kDa as revealed by both SDS-PAGE and immunoblot analyses with anti-SMN, anti-DTx, and anti-TTC antibodies . Like wild-type SMN, purified SDT showed specific binding in vitro to an RG peptide derived from Ewing's sarcoma protein . The fusion protein also bound to cultured primary neurons in amounts similar to those achieved by TTC . Unlike the case with TTC, however, immunolabeling of SDT-treated neurons with anti-TTC and anti-SMN antibodies showed staining restricted to the cell surface . Results from cytotoxicity studies in which the DTx catalytic domain of SDT was used as a reporter protein for internalization and membrane translocation activity suggest that the SMN moiety of the fusion protein is interfering with one or both of these processes . While these studies indicate that SDT may not be useful for SMA therapy, the use of the TTC:DTx fusion construct to deliver other passenger proteins to the neuronal cytosol should not be ruled out. FEBS Lett, 2003 Dec 4, 555(2), 263 - 7 Mechanistic studies of the SufS-SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE; Ollagnier-de-Choudens S et al.; SufS is a cysteine desulfurase of the suf operon shown to be involved in iron-sulfur cluster biosynthesis under iron limitation and oxidative stress conditions . The enzyme catalyzes the conversion of L-cysteine to L-alanine and sulfide through the intermediate formation of a protein-bound cysteine persulfide in the active site . SufE, another component of the suf operon, has been previously shown to bind tightly to SufS and to drastically stimulate its cysteine desulfurase activity . Working with Escherichia coli proteins, we here demonstrate that a conserved cysteine residue in SufE at position 51 is essential for the SufS/SufE cysteine desulfurase activity . Mass spectrometry has been used to demonstrate (i) . the ability of SufE to bind sulfur atoms on its cysteine 51 and (ii) . the direct transfer of the sulfur atom from the cysteine persulfide of SufS to SufE . A reaction mechanism is proposed for this novel two-component cysteine desulfurase. FEBS Lett, 2003 Dec 4, 555(2), 229 - 35 Outer membrane protein A of Escherichia coli forms temperature-sensitive channels in planar lipid bilayers; Zakharian E et al.; The temperature dependence of single-channel conductance and open probability for outer membrane protein A (OmpA) of Escherichia coli were examined in planar lipid bilayers . OmpA formed two interconvertible conductance states, small channels, 36-140 pS, between 15 and 37 degrees C, and large channels, 115-373 pS, between 21 and 39 degrees C . Increasing temperatures had strong effects on open probabilities and on the ratio of large to small channels, particularly between 22 and 34 degrees C, which effected sharp increases in average conductance . The data infer that OmpA is a flexible temperature-sensitive protein that exists as a small pore structure at lower temperatures, but refolds into a large pore at higher temperatures. Mutat Res, 2003 Nov, 544(2-3), 143 - 57 New insights on how nucleotide excision repair could remove DNA adducts induced by chemotherapeutic agents and psoralens plus UV-A (PUVA) in Escherichia coli cells; Lage C et al.; Chemotherapeutic agents such as mitomycin C or nitrogen mustards induce DNA inter-strand cross-links (ICL) and are highly toxic, thus constituting an useful tool to treat some human degenerative diseases, such as cancer . Additionally, psoralens plus UV-A (PUVA), which also induce ICL, find use in treatment of patients afflicted with psoriasis and vitiligo . The repair of DNA ICL generated by different molecules involves a number of multi-step DNA repair pathways . In bacteria, as in eukaryotic cells, if DNA ICL are not tolerated or repaired via nucleotide excision repair (NER), homologous recombination or translesion synthesis pathways, these DNA lesions may lead to mutations and cell death . Herein, we bring new insights to the role of Escherichia coli nucleotide excision repair genes uvrA, uvrB and uvrC in the repair of DNA damage induced by some chemotherapeutic agents and psoralen derivatives plus UV-A . These new observations point to a novel role for the UvrB protein, independent of its previously described role in the Uvr(A)BC complex, which could be specific for repair of monoadducts, intra-strand biadducts and/or ICL. Biochim Biophys Acta, 2003 Dec 1, 1652(2), 107 - 14 Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi; Pinto AP et al.; Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi . Evidences have shown that Ca(2+) may play an important regulatory role in trypanosomatid flagellar mobility . In these parasites, the response of the cell to variations of Ca(2+) levels is determined by a variety of calcium-modulated proteins . Starting from T . cruzi cDNA lambdagt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected . rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment . In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography . Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca(2+) binding . The Ca(2+) binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy . Fluorescence titration exhibited two classes of Ca(2+)-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K(a) of 3.3+/-0.5 (10(6)) and 1.9+/-0.2 (10(4)) M(-1) . Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca(2+)-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor. Biochim Biophys Acta, 2003 Dec 1, 1652(2), 83 - 90 Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus; Leach RN et al.; A polypeptide corresponding to the full-length C-terminal cytoplasmic domain of a G-protein-regulated inwardly rectifying potassium channel (Kir3.1) bearing a hexahistidine (His6) tag was produced by DNA recombinant overexpression techniques in Escherichia coli . This permitted the isolation of approximately 5 mg of pure protein per liter of bacterial culture . Further purification by size exclusion chromatography (SEC) of the C-terminal domain revealed that it exists predominantly as a dimer . The secondary structure was estimated using circular dichroism measurements that indicated the presence of approximately 35% beta-sheet and approximately 15% alpha-helix . G-protein betagamma subunits incubated with His-tagged Kir3.1 C-terminal domain, bound to immobilized metal affinity chromatography (IMAC) resin, copurified with the peak of specifically eluted recombinant protein . These observations demonstrate that full-length Kir3.1 C-terminus can be purified in a stable conformation capable of binding proteins known to activate Kir3 channels and may contain elements involved in channel assembly. J Mol Biol, 2003 Dec 12, 334(5), 1101 - 15 Computational design and characterization of a monomeric helical dinuclear metalloprotein; Calhoun JR et al.; The de novo design of di-iron proteins is an important step towards understanding the diversity of function among this complex family of metalloenzymes . Previous designs of due ferro (DF) proteins have resulted in tetrameric and dimeric four-helix bundles having crystallographically well-defined structures and active-site geometries . Here, the design and characterization of DFsc, a 114 residue monomeric four-helix bundle, is presented . The backbone was modeled using previous oligomeric structures and appropriate inter-helical turns . The identities of 26 residues were predetermined, including the primary and secondary ligands in the active site, residues involved in active site accessibility, and the gamma beta gamma beta turn between helices 2 and 3 . The remaining 88 amino acid residues were determined using statistical computer aided design, which is based upon a recent statistical theory of protein sequences . Rather than sampling sequences, the theory directly provides the site-specific amino acid probabilities, which are then used to guide sequence design . The resulting sequence (DFsc) expresses well in Escherichia coli and is highly soluble . Sedimentation studies confirm that the protein is monomeric in solution . Circular dichroism spectra are consistent with the helical content of the target structure . The protein is structured in both the apo and the holo forms, with the metal-bound form exhibiting increased stability . DFsc stoichiometrically binds a variety of divalent metal ions, including Zn(II), Co(II), Fe(II), and Mn(II), with micromolar affinities . 15N HSQC NMR spectra of both the apo and Zn(II) proteins reveal excellent dispersion with evidence of a significant structural change upon metal binding . DFsc is then a realization of complete de novo design, where backbone structure, activity, and sequence are specified in the design process. J Mol Biol, 2003 Dec 12, 334(5), 1063 - 76 Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP; Zoetewey DL et al.; Bacteria respond to increasing medium osmolality by accumulating organic solutes that are compatible with cellular functions . Transporter ProP of Escherichia coli, a proton symporter and a member of the major facilitator superfamily, senses osmotic shifts and responds by importing osmolytes such as glycine betaine . ProP contains a cytoplasmic, C-terminal extension that is essential for its activity . A peptide corresponding to the C-terminal extension of ProP forms a homodimeric alpha-helical coiled-coil even though some of its heptad a positions are not occupied by hydrophobic amino acid residues . Unexpectedly, amino acid replacement R488I, occurring at a heptad a position, destabilized the coiled-coil formed by the ProP peptide and attenuated the response of the intact transporter to osmotic upshifts in vivo . Thus, ProP was proposed to dimerize via an antiparallel coiled-coil . We used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the synthetic peptide corresponding to residues 468-497 of ProP . This region did form an antiparallel coil-coil in which critical residue R488 specifies the antiparallel coiled-coil orientation by forming stabilizing salt-bridges . Charged residues (both acidic and basic) are clustered on the c/g surface of the coiled-coil whereas polar residues are distributed on the b/e surface . This causes the structure to be bent, in contrast to other known antiparallel coiled-coils (those from the hepatitis delta antigen (PDB ID code 1A92) and the bovine F(1) ATPase inhibitor protein (PDB ID code 1HF9)) . The coiled-coil and its possible importance for osmosensing are discussed. J Mol Biol, 2003 Dec 12, 334(5), 967 - 78 Gene conversion in transposition of Escherichia coli element IS30; Olasz F et al.; The mobile element IS30 has dual target specificity, since it can integrate adjacent to the inverted repeat (IR) of another IS30 copy or into hot-spot sequences characterized by a well-defined consensus showing no similarity to the ends of the element . The result of such integrations into these targets is different, as gene conversion events take place frequently during insertion next to an IR end, while this phenomenon has never been observed in targeting hot-spot sequences . Conversion events in IR-targeting cannot be explained exclusively by the activity of the transposase, but suggest the involvement of the homologous recombination and repair machinery of the host cell . Here, we show that the homology between the donor and target sequences is required for conversion and the starting point of the process is the site of integration . The frequency of conversion depends on the distance of mutations from the end of the targeted element . Remarkable bias is found in the role of donor and target DNA, since generally the donor sequence is converted depending on the target . Conversion was shown to occur also without formation of transposition products . All these data are consistent with the idea of the establishment, migration and resolution of a Holliday-like cruciform structure, which can be responsible for conversion events . To explain the variety of conversion products in IR-targeting, a molecular model has been proposed and discussed. J Mol Biol, 2003 Dec 12, 334(5), 933 - 47 DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori; Zawilak A et al.; The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized . The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane . The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies . DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes . In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments . As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM) . Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity . Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all. Bioorg Med Chem Lett, 2003 Dec 15, 13(24), 4515 - 8 An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo{(4,5)-b}pyridine; Mitsui T et al.; To develop unnatural base pairs that function in replication, we designed 4-propynylpyrrole-2-carbaldehyde (designated as Pa') and synthesized the nucleoside derivatives of Pa' . The base pairing of Pa' with the partner, 9-methylimidazo{(4,5)-b}pyridine (Q), was compared to that of pyrrole-2-carbaldehyde (Pa), which was previously developed as a specific pairing partner of Q . The thermal stability of a DNA duplex containing the Q-Pa' pair and the incorporation efficiency of the Pa' substrate (dPa'TP) into DNA opposite Q by the Klenow fragment of Escherichia coli DNA polymerase I were improved, in comparison with those of the Q-Pa pair . These improvements result from the increased hydrophobicity and stacking stability of Pa' by the introduction of the propynyl group to Pa, providing valuable information for the further development of unnatural base pairs toward the expansion of the genetic alphabet. J Struct Biol, 2003 Oct-Nov, 144(1-2), 162 - 71 Issues of resolution and polymorphism in single-particle reconstruction; Yang S et al.; Three-dimensional reconstruction from electron microscopic (EM) images of isolated macromolecular complexes is being employed by many laboratories . This approach is extremely powerful and continues to improve in resolution . In the absence of stereochemical constraints that can be used to assess the quality of a reconstruction, as exist in X-ray crystallography, several other measures have typically been used . A very useful assessment of quality can be made in the comparison between the projections of the three-dimensional reconstruction and averages generated from classes of images . The main quantitative measure has been that of resolution statistics, typically based upon Fourier shell correlations . We show, using only simulated noise for images, that impressive resolution statistics are generated that can even extend the apparent resolution of the starting model . When truly independent reconstructions are generated starting from different initial models, however, such artefacts are not possible . We also show, using real images of DnaB rings, that in the presence of polymorphism artefactual reconstructions can be generated whose projections match class averages . These averages, however, are themselves artefactual as they involve heterogeneous images . The issues presented here need to be considered when single-particle EM reconstructions are evaluated. J Struct Biol, 2003 Oct-Nov, 144(1-2), 95 - 103 Fast 3D motif search of EM density maps using a locally normalized cross-correlation function; Rath BK et al.; Three-dimensional motif search is becoming increasingly important both in the search for molecular signatures within a tomographic reconstruction, at low resolution, and in the search for atomic structures within high-resolution cryo-EM maps of macromolecular complexes . The present work describes the implementation of a fast local correlation algorithm suitable for template matching in the SPIDER environment . Two examples are given, one in each of the areas of application: (i) . within a 7.8A single-particle reconstruction of the Escherichia coli ribosome, four proteins and one RNA structure were located with high accuracy; (ii) . within a cryo-tomogram of sarcoplasmic reticulum vesicles, ryanodine receptors were located in positions that agreed with expert knowledge. J Struct Biol, 2003 Dec, 144(3), 313 - 9 Crystal structure of SecB from Escherichia coli; Dekker C et al.; The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA . The crystal structure of SecB from E . coli has been solved to 2.35 A resolution . The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site . Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers . Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop . The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr . The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation. Comput Biol Chem, 2003 Oct, 27(4-5), 481 - 95 Investigating protein domain combinations in complete proteomes; Nikitin F et al.; Protein-related information is more accumulated rather than reduced to a synthetic view . Itemising properties of protein sequences is informative, so is the list of ingredients to do some cooking, but without a recipe, that is, quantification and chronology, understanding is incomplete . If the goal of accumulating information is to discover or reveal the function and related biochemical mechanisms, information has to be weighed and ordered . As a guideline, the weight of a piece of information should reflect how often it consistently occurs in various contexts . We propose a common sense approach to quantify and put data and information into perspective . Complete bacterial proteomes are individually mapped with the Pfam-A database of domains and protein family signatures in an attempt to assess the modularity of proteins at the level of a single proteome and the implications of a modular description of proteins for a functional interpretation . Poorly annotated proteins in the most documented bacteria (E . coli and B . subtilis) were considered in an attempt to formulate hypothesis on the basis of domain/module content. DNA Repair (Amst), 2003 Dec 9, 2(12), 1449 - 63 Arsenite and its biomethylated metabolites interfere with the formation and repair of stable BPDE-induced DNA adducts in human cells and impair XPAzf and Fpg; Schwerdtle T et al.; The underlying mechanisms of arsenic carcinogenicity are only poorly understood and especially the role of biomethylation is still a matter of debate . Besides the induction of oxidative DNA damage the interference with DNA repair processes have been proposed to contribute to arsenic-induced carcinogenicity . Within the present study the effects of arsenite and its mono- and dimethylated trivalent and pentavalent metabolites on BPDE-induced DNA adduct formation and repair has been investigated and compared in cultured human lung cells . Whereas only arsenite and MMA(III) increased BPDE-DNA adduct formation, arsenite (>/=5 microM), the trivalent (>/=2.5 microM) and the pentavalent (>/=250 microM) metabolites diminished their repair at non-cytotoxic concentrations . As potential molecular targets, interactions with the zinc finger domain of the human XPA protein (XPAzf) and the Escherichia coli zinc finger protein Fpg, involved in NER and BER, respectively, have been investigated . All trivalent arsenicals were able to release zinc from XPAzf; furthermore, MMA(III) and DMA(III) inhibited the activity of isolated Fpg . Altogether the results suggest that besides arsenite, especially the trivalent methylated metabolites may contribute to diminished NER at low concentrations. DNA Repair (Amst), 2003 Dec 9, 2(12), 1419 - 27 Expression of Deinococcus radiodurans PprI enhances the radioresistance of Escherichia coli; Gao G et al.; PprI, a newly identified gene switch responsible for extreme radioresistance of Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair and protection pathways in response to radiation stress {Biochem . Biophy . Res . Commun . 306 (2003) 354} . To evaluate whether PprI also functions in the radioresistance in other organisms, D . radiodurans PprI protein (Deira-PprI) was expressed in Escherichia coli . The complemented E . coli strain showed an increase of approximately 1.6-fold radioresistance with a high dose of gamma irradiation . Immunoblotting assays showed that the expression of Deira-PprI in E . coli resulted in a significant increase in RecA protein expression following high dose ionizing radiation . The expression of Deira-PprI protein also significantly enhanced the scavenging ability of free radicals by inducing the enzymatic activity of KatG . These results indicate that exogenous expression of Deira-PprI promotes DNA repair and protection pathways and enhances the radioresistance of E . coli. Chin Med J (Engl), 2003 Nov, 116(11), 1678 - 82 Protective effects of alpha 1-antitrypsin on acute lung injury in rabbits induced by endotoxin; Jie Z et al.; OBJECTIVE: To investigate whether pretreatment with alpha(1)-antitrypsin (AAT) can attenuate acute lung injury (ALI) in rabbits induced with endotoxin . METHODS: Thirty-two healthy adult New Zealand rabbits were anaesthetized, tracheotomized and mechanically ventilated . They were then randomly divided into four groups (n = 8): (1) Infusion of Escherichia coli endotoxin {Lipopolysaccharide (LPS) 500 microg/kg} without AAT (Group LPS) . (2) Infusion of AAT 120 mg/kg at 15 minutes after LPS (Group LAV) . (3) Infusion of AAT 120 mg/kg without endotoxin (Group AAT) . (4) Infusion of saline 4 ml/kg as control (Group NS) . Arterial blood gases, peripheral leukocyte counts and airway pressure were recorded every hour for eight hours . Physiologic intrapulmonary shunting (Qs/Qt) was measured every four hours . After eight hours, blood samples were collected for measurement of plasma concentration and activity of AAT . Then, the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected for measurement of concentrations of total protein (TP), interleukin-8 (IL-8), tumor necrosis factor (TNF alpha, the activities of NE and AAT, total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) . In addition, the wet-to-dry lung weight ratio (W/D) was measured . RESULTS: The infusion of endotoxin induced decreases in arterial oxygen pressure (PaO(2)), peripheral leukocyte counts, total respiratory compliance (TLC) and the increases in peak pressure (P(peak)), Qs/Qt compared with the baseline values (P < 0.05) . The increased plasma concentration but reduced activity of AAT was also found in contrast to that in Group NS (P < 0.05) . In the BALF, the activity of AAT, TPL, DSPC/TPL were lower than those in Group NS (P < 0.05), but the concentrations of albumin, IL-8, TNF alpha, the activity of NE and the ratio of W/D were higher than those in Group NS (P < 0.05) . The pretreatment of AAT attenuated the deterioration of oxygenation, the reduction of compliance and the deterioration of other physiological and biochemical parameters mentioned above . CONCLUSION: Pretreatment with AAT could attenuate endotoxin-induced lung injury in rabbits . Those beneficial effects of AAT might be due, in part, to reduction in the levels of mediators that could activate neutrophils, in addition to the direct inhibitory effect on neutrophil elastase. Biochem Soc Trans, 2003 Dec, 31(Pt 6), 1343 - 8 Glyoxalase I--structure, function and a critical role in the enzymatic defence against glycation; Thornalley PJ; Glyoxalase I is part of the glyoxalase system present in the cytosol of cells . The glyoxalase system catalyses the conversion of reactive, acyclic alpha-oxoaldehydes into the corresponding alpha-hydroxyacids . Glyoxalase I catalyses the isomerization of the hemithioacetal, formed spontaneously from alpha-oxoaldehyde and GSH, to S -2-hydroxyacylglutathione derivatives {RCOCH(OH)-SG-->RCH(OH)CO-SG}, and in so doing decreases the steady-state concentrations of physiological alpha-oxoaldehydes and associated glycation reactions . Physiological substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclic alpha-oxoaldehydes . Human glyoxalase I is a dimeric Zn(2+) metalloenzyme of molecular mass 42 kDa . Glyoxalase I from Escherichia coli is a Ni(2+) metalloenzyme . The crystal structures of human and E . coli glyoxalase I have been determined to 1.7 and 1.5 A resolution . The Zn(2+) site comprises two structurally equivalent residues from each domain--Gln-33A, Glu-99A, His-126B, Glu-172B and two water molecules . The Ni(2+) binding site comprises His-5A, Glu-56A, His-74B, Glu-122B and two water molecules . The catalytic reaction involves base-catalysed shielded-proton transfer from C-1 to C-2 of the hemithioacetal to form an ene-diol intermediate and rapid ketonization to the thioester product . R - and S-enantiomers of the hemithioacetal are bound in the active site, displacing the water molecules in the metal ion primary co-ordination shell . It has been proposed that Glu-172 is the catalytic base for the S-substrate enantiomer and Glu-99 the catalytic base for the R-substrate enantiomer; Glu-172 then reprotonates the ene-diol stereospecifically to form the R-2-hydroxyacylglutathione product . By analogy with the human enzyme, Glu-56 and Glu-122 may be the bases involved in the catalytic mechanism of E . coli glyoxalase I . The suppression of alpha-oxoaldehyde-mediated glycation by glyoxalase I is particularly important in diabetes and uraemia, where alpha-oxoaldehyde concentrations are increased . Decreased glyoxalase I activity in situ due to the aging process and oxidative stress results in increased glycation and tissue damage . Inhibition of glyoxalase I pharmacologically with specific inhibitors leads to the accumulation of alpha-oxoaldehydes to cytotoxic levels; cell-permeable glyoxalase I inhibitors are antitumour and antimalarial agents . Glyoxalase I has a critical role in the prevention of glycation reactions mediated by methylglyoxal, glyoxal and other alpha-oxoaldehydes in vivo. Biochemistry (Mosc), 2003 Nov, 68(11), 1195 - 9 Active dimeric form of inorganic pyrophosphatase from Escherichia coli; Vainonen YP et al.; A dimeric form can be obtained from native hexameric Escherichia coli inorganic pyrophosphatase (E-PPase) by destroying the hydrophobic intersubunit contacts, and it has been shown earlier to consist of the subunits of different trimers . The present paper is devoted to the kinetic characterization of such a "double-decked" dimer obtained by the dissociation of either the native enzyme or the mutant variant Glu145Gln . The dimeric form of the native inorganic pyrophosphatase was shown to retain high catalytic efficiency that is in sharp contrast to the dimers obtained as a result of the mutations at the intertrimeric interface . The dimeric enzymes described in the present paper, however, have lost the regulatory properties, in contrast to the hexameric and trimeric forms of the enzyme. Physiol Res, 2003, 52(6), 789 - 96 Several functions of immune cells in mice changed by oxidative stress caused by endotoxin; Victor VM et al.; We have studied natural killer (NK) activity, lymphoproliferative response, the release of several cytokines (IL-2, TNF alpha and IL-1 beta) and the ROS production in peritoneal leukocytes obtained 0, 2, 4, 12 and 24 h after lipopolysaccharide (LPS) injection . Lethal septic shock (100 % mortality occurred at 30 h after LPS administration) was caused in female BALB/c mice by intraperitoneal injection of 100 mg/kg of E . coli LPS . Cytotoxicity and lymphoproliferation assay were preformed together with the measurement of IL-1 beta, IL-2 and TNF alpha production, and quantification of ROS . Natural killer activity, spontaneous lymphoproliferative response, IL-2, TNF alpha, IL-beta release and ROS production were increased after LPS injection . In conclusions, ROS and proinflammatory mediators produced by immune cells in response to LPS are involved in the oxidative stress of endotoxic shock . This oxidative state alters some functional characteristics of leukocytes (proliferation and NK activity). Biochemistry, 2003 Dec 9, 42(48), 14306 - 17 Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL; Powl AM et al.; Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer . Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex . The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues . We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants . In vivo cell viability assays in E . coli show that introduction of the Trp residues does not block function of the channels . The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes . Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid . Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL . Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca . 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure . Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body . It is concluded that MscL distorts to match changes in bilayer thickness . The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1 . Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids . Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine. Biochemistry, 2003 Dec 9, 42(48), 14242 - 8 Structure and function of the middle domain of ClpB from Escherichia coli; Kedzierska S et al.; ClpB belongs to the Hsp100/Clp ATPase family . Whereas a homologue of ClpB, ClpA, interacts with and stimulates the peptidase ClpP, ClpB does not associate with peptidases and instead cooperates with DnaK/DnaJ/GrpE in an efficient reactivation of severely aggregated proteins . The major difference between ClpA and ClpB is located in the middle sequence region (MD) that is much longer in ClpB than in ClpA and contains several segments of coiled-coil-like heptad repeats . The function of MD is unknown . We purified the isolated MD fragment of ClpB from Escherichia coli (residues 410-570) . Circular dichroism (CD) detected a high population of alpha-helical structure in MD . Temperature-induced changes in CD showed that MD is a thermodynamically stable folding domain . Sedimentation equilibrium showed that MD is monomeric in solution . We produced four truncated variants of ClpB with deletions of the following heptad-repeat-containing regions in MD: 417-455, 456-498, 496-530, and 531-569 . We found that the removal of each heptad-repeat region within MD strongly inhibited the oligomerization of ClpB, which produced low ATPase activity of the truncated ClpB variants as well as their low chaperone activity in vivo . Only one ClpB variant (Delta417-455) could partially complement the growth defect of the clpB-null E . coli strain at 50 degrees C . Our results show that heptad repeats in MD play an important role in stabilization of the active oligomeric form of ClpB . The heptad repeats are likely involved in stabilization of an intra-MD helical bundle rather than an intersubunit coiled coil. Biochemistry, 2003 Dec 9, 42(48), 14225 - 33 Effects of site-directed mutations on heme reduction in Escherichia coli nitrate reductase A by menaquinol: a stopped-flow study; Zhao Z et al.; We have studied the effects of site-directed mutations in Escherichia coli nitrate reductase A (NarGHI) on heme reduction by a menaquinol analogue (menadiol) using the stopped-flow method . For NarGHI(H66Y) and NarGHI(H187Y), both lacking heme b(L) but having heme b(H), the heme reduction by menadiol is abolished . For NarGHI(H56R) and NarGHI(H205Y), both without heme b(H) but with heme b(L), a smaller and slower heme reduction compared to that of the wild-type enzyme is observed . These results indicate that electrons from menadiol oxidation are transferred initially to heme b(L) . A transient species, likely to be associated with a semiquinone radical anion, was generated not only on reduction of the wild-type enzyme as observed previously (1) but also on reduction of NarGHI(H56R) and NarGHI(H205Y) . The inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide and stigmatellin both have significant effects on the reduction kinetics of NarGHI(H56R) and NarGHI(H205Y) . We have also investigated the reoxidation of menadiol-reduced heme by nitrate in the mutants . Compared to the wild type, no significant heme reoxidation is observed for NarGHI(H56R) and NarGHI(H205Y) . This result indicates that a single mutation removing heme b(H) blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH. J Med Chem, 2003 Dec 4, 46(25), 5533 - 45 Synthesis and evaluation of nitroheterocyclic carbamate prodrugs for use with nitroreductase-mediated gene-directed enzyme prodrug therapy; Hay MP et al.; A variety of nitroheterocyclic carbamate prodrugs of phenylenediamine mustard and 5-amino-1-(chloromethyl)-3-{(5,6,7-trimethoxyindol-2-yl)carbonyl}-1,2-dihydro-3H-benz{e}indoline (amino-seco-CBI-TMI), covering a wide range of reduction potential, were prepared and evaluated for use in gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from Escherichia coli B . The carbamate prodrugs and corresponding amine effectors were tested in a cell line panel comprising parental and NTR-transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79(puro)/V79-NTR(puro)), and murine (EMT6/EMT6-NTR(puro)) cell line pairs and were compared with the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard . The 1-methyl-2-nitroimidazol-5-ylmethyl carbamate of phenylenediamine mustard was metabolized rapidly by EMT6-NTR(neo) but not EMT6 cells, demonstrating that it is an efficient substrates for NTR . Despite this, the carbamates of phenylenediamine mustards show relatively low differential cytotoxicity for NTR+ve cells in IC(50) assays, apparently because they retain sufficient alkylating reactivity that most of the prodrug reacts with nucleophiles during the drug exposure period . In contrast, the corresponding amino-seco-CBI-TMI prodrugs were less efficient NTR substrates but had greater chemical stability, were more potent, and showed substantial NTR-ve/NTR+ve ratios in the cell line panel, with ratios of 15-100-fold for the 1-methyl-2-nitro-1H-imidazol-5-ylmethyl and 1-methyl-5-nitro-1H-imidazol-2-ylmethyl carbamates of amino-seco-CBI-TMI . The activity of these two prodrugs was evaluated against NTR-expressing EMT6 tumors comprising ca . 10% NTR+ve cells . Small but not statistically significant killing of NTR+ve cells was observed, with no effect against NTR-ve target cells . The lack of activity against NTR+ve cells in tumors, despite potent and selective activity in culture, indicates that pharmacokinetic optimization will be required if in vivo efficacy against solid tumors is to be achieved with this new class of NTR prodrugs. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2003 Oct, 25(5), 557 - 62 {Cloning and expression of human single-chain Fv antibody against amyloid beta peptide involved in Alzheimer's disease}; Cai J et al.; OBJECTIVE: To screen out the specific antibody clones against amyloid beta peptide 40, and clone the antibody gene and express it in a bacterial system, so as to provide a solid basis for novel diagnostic and therapeutic methods for Alzheimer's Disease . METHODS: beta amyloid peptide 40 was bound on the solid surface of Nunc plates as antigen to screen the binding clones from a phage-display human single-chain Fv antibody library . After five rounds of bio-panning, the host E . coli TG1 was infected with eluted filamentous phage from the last turn of selection . 55 well-separated colonies were picked randomly from the plates and the specific positive clones were identified by ELISA test . The single-chain Fv antibody gene was sequenced and their amino acids sequence was deduced . The scFv antibody gene was sub-cloned into a protokayotic expression vector pGEX-6P-1 and transformed into bacteria strain BL21 to express the glutathione-S-transferase (GST) fusion single-chain antibody . RESULTS: ELISA test showed that 33 of the 55 clones could bind amyloid beta peptide 40 and 10 of the 33 clones could be inhibited by amyloid beta peptide 40 itself to below 50% of its original binding activities . Five of the 10 clones could also be inhibited by amyloid beta peptide 1-16 to the same level, which meant that the binding epitope of the antibody from the 5 clones was between first to sixteenth amino acids at amino-end of amyloid beta peptide 40 . DNA sequencing data demonstrated that the gene of the single-chain antibody specifically against amyloid beta peptide 40 was consisted of 768 bp and the deduced amino acids sequence confirmed its typical antibody structure . The complement determinant regions and framework regions were discriminated empirically . After cloning the antibody gene into a protokayotic system, the GST fusion antibody was expressed as the expected size . CONCLUSIONS: After five rounds of bio-panning and subsequently serial ELISA testing, the specific antibody clones against amyloid beta peptide 40 were screened out successfully . The antibody gene DNA sequence and amino acids sequence were analyzed and confirmed . The fusion antibody was expressed as expected in the bacterial system. J Struct Funct Genomics, 2003, 4(2-3), 167 - 77 From protein structure to biochemical function? Laskowski RA, Watson JD, Thornton JM. Here we describe various methods currently under development aimed at identifying a protein's function from its three-dimensional structure . We are combining a number of these methods to create a pipeline of applications, called ProFunc, which will take a given 3D structure, run all the applications on it and compile and summarise the results obtained . The aim is to provide a best guess as to the protein's function from the evidence provided by the different methods . Here we present three examples, using structures solved by the Midwest Center for Structural Genomics consortium, illustrating the strengths and weaknesses of current approaches. J Struct Funct Genomics, 2003, 4(2-3), 137 - 9 Structural genomics efforts at the Chinese Academy of Sciences and Peking University; Gong WM et al.; Structural genomics efforts at the Chinese Academy of Sciences and Peking University are reported in this article . The major targets for the structural genomics project are targeted proteins expressed in human hematopoietic stem/progenitor cells, proteins related to blood diseases and other human proteins . Up to now 328 target genes have been constructed in expression vectors . Among them, more than 50% genes have been expressed in Escherichia coli, approximately 25% of the resulting proteins are soluble, and 35 proteins have been purified . Crystallization, data collection and structure determination are continuing . Experiences accumulated during this initial stage are useful for designing and applying high-throughput approaches in structural genomics. J Struct Funct Genomics, 2003, 4(2-3), 47 - 55 Coverage of protein sequence space by current structural genomics targets; O'Toole N et al.; By its purest definition the ultimate goal of structural genomics (SG) is the determination of the structures of all proteins encoded by genomes . Most of these will be obtained by homology modeling using the structures of a set of target proteins for experimental determination . Thanks to the open exchange of SG target information, we are able to analyze the sequences of the current target list to evaluate the extent of its coverage of protein sequence space . The presence of homologous sequences currently either in the Protein Data Bank (PDB) or among SG targets has been determined for each of the protein sequences in several organisms . In this way we are able to evaluate the coverage by existing or targeted structural data for the non-membranous parts of entire proteomes . For small bacterial proteomes such as that of H . influenzae almost all proteins have homologous sequences among SG targets or in the PDB . There is significantly lower coverage for more complex organisms, such as C . elegans . We have mapped the SG target list onto the ProtoMap clustering of protein sequences . Clusters occupied by SG targets represent over 150,000 protein sequences, which is approximately 44% of the total protein sequences classified by ProtoMap . The mapping of SG targets also enables an evaluation of the degree of overlap within the target list . An SG target typically occupies a ProtoMap cluster with more than six other homologous targets. J Biol Inorg Chem, 2004 Jan, 9(1), 90 - 5 Epub 2003 Nov 29. Sequential reconstitution of copper sites in the multicopper oxidase CueO; Galli I et al.; CueO belongs to the family of multicopper oxidases which are characterized by the presence of multiple copper-binding sites with different structural and functional properties . These enzymes share the ability to couple the one-electron oxidation of substrate to reduction of oxygen to water by way of a functional unit composed of a mononuclear type 1 blue copper site, which is the entry site for electrons, and of a trinuclear copper cluster formed by type 2 and binuclear type 3 sites, where oxygen binding and reduction take place . The mechanism of copper incorporation in CueO has been investigated by optical and EPR spectroscopy . The results indicate unambiguously that the process is sequential, with type 1 copper being the first to be reconstituted, followed by type 2 and type 3 sites. Cancer Immunol Immunother, 2004 Jun, 53(6), 497 - 509 Epub 2003 Nov 25. A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells; Hayashi H et al.; PURPOSE: In the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach . Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems . METHODS: We have produced an anti-epidermal growth-factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E . coli) expression system with refolding method . The Ex3 diabody targets lymphokine-activated killer cells with a T-cell phenotype (T-LAK cells) to EGFR positive bile duct carcinoma cells with dramatic enhancement of cytotoxicity in vitro . This specific killing of EGFR-positive cells was completely inhibited by parental mAb IgGs directed to EGFR and the CD3 antigen . RESULTS: When T-LAK cells were cultured with EGFR-positive tumor cells in the presence of Ex3 diabody, they produced much higher levels of IFN-gamma, GM-CSF, and TNF-alpha than in its absence, this being a possible mechanism underlying specific antitumor activity . The Ex3 diabody showed good stability when tested at 37 degrees C for 48 h, and also markedly inhibited tumor growth of bile duct carcinoma xenografts in severe combined immunodeficient (SCID) mice . When Ex3 diabody (20 microg/mouse) was administrated intravenously, together with T-LAK cells and interleukin-2 (IL-2), complete cure of tumors were observed in three of six mice, and the other three showed marked retardation of tumor growth . CONCLUSION: The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2322 - 4 Epub 2003 Nov 27. Crystallization and preliminary X-ray analysis of the glycogen synthase from Pyrococcus abyssi; Horcajada C et al.; Glycogen synthase catalyzes the transfer of glucosyl residues from ADP- or UDP-glucose to the non-reducing end of a growing alpha-1,4-glucan chain . To date, no crystallographic structure of an animal/fungal glycogen synthase (family 3 of the glycosyl transferases) or a bacterial/plant glycogen/starch synthase (family 5) has been reported . This paper describes the recombinant expression, crystallization and preliminary X-ray analysis of the glycogen synthase from the hyperthermophilic archaeon Pyrococcus abyssi, the smallest enzyme of the members of families 3 and 5 of the glycosyl transferases . Crystals from this protein and from its selenomethionyl variant were grown in 100 mM sodium citrate pH 5.6 containing 20% PEG and 20% dioxane by the hanging-drop vapour-diffusion method at 293 K . The crystals, which grew as thin needles, diffracted to 3.5 A resolution and belong to space group C2, with unit-cell parameters a = 202, b = 73, c = 149 A, beta = 131 degrees . The crystallographic and biochemical data are consistent with either a dimer or a tetramer in the crystal asymmetric unit and a volume solvent content of 70 or 39%, respectively. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2251 - 3 Epub 2003 Nov 27. Crystallization and preliminary crystallographic analysis of the extracellular fragment of FcalphaRI/CD89; Yang M et al.; Crystals of the extracellular fragment of FcalphaRI/CD89 have been grown at 291 K using PEG 8000 as precipitant . The diffraction pattern of the selenomethionine (SeMet) derivative crystal extended to 2.1 A resolution at SPring-8, Japan . The crystals belong to space group C222(1), with unit-cell parameters a = 59.0, b = 69.4, c = 106.3 A, alpha = beta = gamma = 90 degrees . The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.12 A(3) Da(-1) and a solvent content of 60.2% by volume . A full set of X-ray diffraction data were collected to 2.1 A from an SeMet-derivative crystal. J Bacteriol, 2003 Dec, 185(24), 7053 - 67 Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts; Hua Q et al.; The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats . The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on {U-(13)C}glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose . Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency . Furthermore, additional, unexpected flux responses to the knockout mutations were observed . Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E . coli . The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain . Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions. J Biol Chem, 2004 Feb 6, 279(6), 4250 - 9 Epub 2003 Nov 25. Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase; Liu L et al.; It has been proposed that the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A . E., Williams, K . M., and Guengerich, F . P . (2002) J . Biol . Chem . 277, 37920-37928) . Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes . The order of reaction determined was Gua>Thy>Cyt>Ade . Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct . The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions . The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct . Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination . Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s). Biochimie, 2003 Oct, 85(10), 1033 - 9 cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata; Yang Y et al.; Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology . In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle . The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins . Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4 . The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography . The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate . Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins . Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C. Dev Comp Immunol, 2004 Mar, 28(3), 229 - 37 Production and mechanism of secretion of interleukin-1beta from the marine fish gilthead seabream; Pelegrin P et al.; Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion . Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors . Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site . We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta) . The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor . More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip . In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP . Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor. Biochim Biophys Acta, 2003 Dec 5, 1624(1-3), 6 - 10 FT-IR study of heterologous protein expression in recombinant Escherichia coli strains; Ami D et al.; Two recombinant Escherichia coli strains expressing different levels of an interferon fusion protein as inclusion bodies have been studied by Fourier transform infrared (FT-IR) microspectroscopy . A marker band at 1628 cm(-1) allowed monitoring of the protein expression by direct analysis of cell pellets in a rapid, non-invasive and quantitative way . The results demonstrate that FT-IR microspectroscopy is a technique of potential biotechnological interest for studying inclusion body formation. Biochem J, 2004 Mar 15, 378(Pt 3), 1047 - 52 Fructoselysine 3-epimerase, an enzyme involved in the metabolism of the unusual Amadori compound psicoselysine in Escherichia coli; Wiame E et al.; The frl (fructoselysine) operon encodes fructoselysine 6-kinase and fructoselysine 6-phosphate deglycase, allowing the conversion of fructoselysine into glucose 6-phosphate and lysine . We now show that a third enzyme encoded by this operon catalyses the metal-dependent reversible interconversion of fructoselysine with its C-3 epimer, psicoselysine . The enzyme can be easily assayed through the formation of tritiated water from {3-3H}fructoselysine . Psicoselysine supports the growth of Escherichia coli, causing the induction of the three enzymes of the frl operon . No growth on fructoselysine or psicoselysine was observed with Tn5 mutants in which the putative transporter (FrlA) or fructoselysine 6-phosphate deglycase (FrlB) had been inactivated, indicating the importance of the frl operon for the metabolism of both substrates . The ability of E . coli to grow on psicoselysine suggests the occurrence of this unusual Amadori compound in Nature. Biochemistry, 2003 Dec 9, 42(48), 14318 - 27 DNA damage and cytotoxicity induced by minor groove binding methyl sulfonate esters; Varadarajan S et al.; Minor groove specific DNA equilibrium binding peptides (lex) based on N-methylpyrrole-carboxamide and/or N-methylimidazolecarboxamide subunits have been modified with an O-methyl sulfonate ester functionality to target DNA methylation in the minor groove at Ade/Thy- and/or Gua/Cyt-rich sequences . HPLC and sequencing gel analyses show that the Me-lex compounds all selectively react with DNA to afford N3-alkyladenine as a major adduct . The formation of the N3-alkyladenine lesions is sequence-dependent based on the equilibrium binding preferences of the different lex peptides . In addition to the reaction at adenine, the molecules designed to target Gua/Cyt sequences also generate lesions at guanine; however, the methylation is not sequence dependent and takes places in the major groove at the N7-position . To determine if and how the level of the different DNA adducts and the sequence selectivity for their formation affects cytotoxicity, the Me-lex analogues were tested in wild type Escherichia coli and in mutant strains defective in base excision repair (tag and/or alkA or apn) . The results demonstrate the importance of 3-methyladenine, and in some cases 3-methylguanine, lesions in cellular toxicity, and the dominant protective role of the DNA glycosylases . There is no evidence that the sequence specificity is related to toxicity. J Nat Prod, 2003 Nov, 66(11), 1512 - 4 New 9-thiocyanatopupukeanane sesquiterpenes from the nudibranch Phyllidia varicosa and its sponge-prey Axinyssa aculeata; Yasman Y et al.; Two new 9-thiocyanatopupukeanane sesquiterpene isomers were isolated as major metabolites from the MeOH extract of the sponge Axinyssa aculeata and from its nudibranch predator Phyllidia varicosa . The presence of the sesquiterpenes was monitored by GC-MS, and the structures were confirmed by both 1D and 2D NMR spectroscopy . The isolated sesquiterpenes were found to be toxic toward brine shrimp at LC(50) of 5 ppm . At a dose level of 20 microg, they were found to be weakly and moderately active against B . subtilis and C . albicans, respectively. Am J Trop Med Hyg, 2003 Oct, 69(4), 406 - 10 Frequency and virulence properties of diarrheagenic Escherichia coli in children with diarrhea in Gabon; Presterl E et al.; To investigate the presence of diarrheagenic Escherichia coli in Lambarene, Gabon, 150 children with diarrhea were screened for enteroaggregative E . coli (EAEC), enterotoxigenic E . coli (ETEC), enteropathogenic E . coli (EPEC), enterohemorrhagic E . coli (EHEC), and enteroinvasive E . coli (EIEC) using polymerase chain reaction and an HEp-2 cell culture techniques . Isolates of EAEC were detected in 57 children, two thirds of them between six months and two years of age, and isolates of ETEC were detected in seven patients . Isolates of EPEC, EHEC, and EIEC were not present in this population . Among the EAEC, the pCVD432 plasmid, a heat stable (ST)-like (ST-like) enterotoxin (EAST), and a plasmid-encoded heat-labile toxin (PET) were detected in 19, 34, and 42 cases, respectively . Detection of pCVD432, EAST, and PET were significantly associated with EAEC identified by the HEp-2 cell assay . Although detected only in 16 patients, the presence of the fimbriae AAF I (aagA) and AAF II (aafA) were more likely to occur in EAEC than in non-EAEC (odds ratio {OR} = 4.1, 95% confidence interval {CI} = 0.5-38.6, and OR = 2.3, 95% CI = 1.0-5.3, respectively) . The EAEC isolates exhibited decreased susceptibility for ampicillin, tetracycline, and trimethoprim. Amyloid, 2003 Sep, 10(3), 147 - 50 Slower clearance of human SAA1.5 in mice: implications for allele specific variation of SAA concentration in human; Yamada T et al.; Serum amyloid A (SAA), the serum precursor of the fibrillar component (AA proteins) in reactive amyloid deposits, is a multigene product . SAA1, the prominent acute phase isotype in serum and also the dominant fibril precursor, has several allelic variants . In Japan, each of the three major alleles (1.1, 1.3 and 1.5) appears with approximately equal frequency . Recent research suggested that allele 1.5 has a positive influence on the serum SAA concentration . To clarify this, in the present study, recombinant human SAA1.1, SAA1.3 and SAA1.5 were produced in an E . coli expression system and those species of reconstituted high density lipoprotein were injected into mice to examine plasma clearance . SAA1.5 disappeared from plasma more slowly than the other two isotypes . This may account for the positive influence of allele 1.5 on the serum SAA concentration. J Infect Dis, 2003 Dec 1, 188(11), 1724 - 9 Epub 2003 Nov 14. Age-specific frequencies of antibodies to Escherichia coli verocytotoxins (Shiga toxins) 1 and 2 among urban and rural populations in southern Ontario; Karmali MA et al.; In 173 urban residents and 232 rural dairy-farm residents (age range, 0-70 years) who were stratified for age, the frequency of antiverocytotoxin 2 antibodies (VT2 Abs) (frequency in urban residents, 46%; frequency in rural residents, 65%) was significantly higher than that of antiverocytotoxin 1 antibodies (VT1 Abs) (frequency in urban residents, 12%; frequency in rural residents, 39%) (P< or =.001) . The frequency of VT2 Abs (93%) was also significantly higher than that of VT1 Abs (50%) in 14 patients with hemolytic uremic syndrome (HUS) associated with verocytotoxin-producing Escherichia coli (VTEC) strains that expressed both toxins . In urban residents, the frequency of both antibodies tended to decrease between the first and the second decades of life, and it then increased until the fifth decade of life, before, in the case of VT2 Abs, decreasing again . This pattern, which inversely reflects the age-related incidence of HUS, is consistent with a role for antiverocytotoxin antibodies in protective immunity . In dairy-farm residents, peak frequencies of antibodies to both toxins occurred during the first decade of life and remained elevated for 3 decades before decreasing, a pattern consistent with frequent exposure to bovine VTEC from an early age. Infect Immun, 2003 Dec, 71(12), 7069 - 78 Disruption of cell polarity by enteropathogenic Escherichia coli enables basolateral membrane proteins to migrate apically and to potentiate physiological consequences; Muza-Moons MM et al.; Enteropathogenic Escherichia coli (EPEC) disrupts the structure and barrier function of host intestinal epithelial tight junctions (TJs) . The impact of EPEC on TJ "fence function," i.e., maintenance of cell polarity, has not been investigated . In polarized cells, proteins such as beta(1)-integrin and Na(+)/K(+) ATPase are restricted to basolateral (BL) membranes . The outer membrane EPEC protein intimin possesses binding sites for the EPEC translocated intimin receptor (Tir) and beta(1)-integrin . Restriction of beta(1)-integrin to BL domains, however, precludes opportunity for interaction . We hypothesize that EPEC perturbs TJ fence function and frees BL proteins such as beta(1)-integrin to migrate to apical (AP) membranes of host cells, thus allowing interactions with bacterial adhesins such as intimin . The aim of this study was to determine whether EPEC alters the polar distribution of BL proteins, in particular beta(1)-integrin, and if such redistribution contributes to pathogenesis . Human intestinal epithelial T84 cells and EPEC strain E2348/69 were used . Selective biotinylation of AP or BL membrane proteins and confocal microscopy showed the presence of beta(1)-integrin and Na(+)/K(+) ATPase on the AP membrane following infection . beta(1)-Integrin antibody afforded no protection against the initial EPEC-induced decrease in transepithelial electrical resistance (TER) but halted the progressive decrease at later time points . While the effects of EPEC on TJ barrier and fence function were Tir dependent, disruption of cell polarity by calcium chelation allowed a tir mutant to be nearly as effective as wild-type EPEC . In contrast, deletion of espD, which renders the type III secretory system ineffective, had no effect on TER even after calcium chelation, suggesting that the putative beta(1)-integrin-intimin interaction serves to provide intimate contact, like that of Tir and intimin, making translocation of effector molecules more efficient . We conclude that the initial alterations of TJ barrier and fence function by EPEC are Tir dependent but that later disruption of cell polarity and accessibility of EPEC to BL membrane proteins, such as beta(1)-integrin, potentiates the physiological perturbations. Infect Immun, 2003 Dec, 71(12), 6799 - 807 Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells; Coats SR et al.; E . coli lipopolysaccharide (LPS) induces cytokine and adhesion molecule expression via the toll-like receptor 4 (TLR4) signaling complex in human endothelial cells . In the present study, we investigated the mechanism by which Porphyromonas gingivalis LPS antagonizes E . coli LPS-dependent activation of human endothelial cells . P . gingivalis LPS at 1 micro g/ml inhibited both E . coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 micro g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1beta) stimulation . P . gingivalis LPS (1 micro g/ml) also blocked both E . coli LPS-dependent and M . tuberculosis HSP60.1-dependent but not IL-1beta-dependent activation of NF-kappaB in human microvascular endothelial (HMEC-1) cells, consistent with antagonism occurring upstream from the TLR/IL-1 receptor adaptor protein, MyD88 . Surprisingly, P . gingivalis LPS weakly but significantly activated NF-kappaB in HMEC-1 cells in the absence of E . coli LPS, and the P . gingivalis LPS-dependent agonism was blocked by transient expression of a dominant negative murine TLR4 . Pretreatment of HUVECs with P . gingivalis LPS did not influence the ability of E . coli LPS to stimulate E-selectin mRNA expression . Taken together, these data provide the first evidence that P . gingivalis LPS-dependent antagonism of E . coli LPS in human endothelial cells likely involves the ability of P . gingivalis LPS to directly compete with E . coli LPS at the TLR4 signaling complex. Infect Immun, 2003 Dec, 71(12), 6766 - 74 Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1; Singh S et al.; Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria . In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1(42)) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities . Biochemical, biophysical, and immunological characterization confirmed that refolded E . coli MSP1(42) was homogeneous and highly immunogenic . In a formulation suitable for human use, rabbit antibodies were raised against refolded E . coli MSP1(42) and tested in vitro in a P . falciparum growth invasion assay . The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P . falciparum MSP1(42) . However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP1(19)) . Vaccination of nonhuman primates with E . coli MSP1(42) in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P . falciparum . The protection correlated with antibody-dependent mechanisms . Thus, this new construct, E . coli MSP1(42), is a viable candidate for human vaccine trials. Infect Immun, 2003 Dec, 71(12), 6747 - 53 Lipopolysaccharide binding protein is an essential component of the innate immune response to Escherichia coli peritonitis in mice; Knapp S et al.; Lipopolysaccharide (LPS) binding protein (LBP) is an acute-phase protein that enhances the responsiveness of immune cells to LPS by virtue of its capacity to transfer LPS to CD14 . To determine the role of LBP in the innate immune response to peritonitis, LBP gene-deficient (LBP(-/-)) and normal wild-type mice were intraperitoneally infected with Escherichia coli, the most common causative pathogen of this disease . LBP was detected at low concentrations in peritoneal fluid of healthy wild-type mice, and the local LBP levels increased rapidly upon induction of peritonitis . LBP(-/-) mice were highly susceptible to E . coli peritonitis, as indicated by accelerated mortality, earlier bacterial dissemination to the blood, impaired bacterial clearance in the peritoneal cavity, and more severe remote organ damage . LBP(-/-) mice displayed diminished early tumor necrosis factor alpha, interleukin-6, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein 2 production and attenuated recruitment of polymorphonuclear leukocytes to the site of infection, indicating that acute inflammation was promoted by LBP . Locally produced LBP is an essential component of an effective innate immune response to E . coli peritonitis. J Biol Chem, 2004 Feb 6, 279(6), 4221 - 33 Epub 2003 Nov 24. S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex; Okada M et al.; Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain . In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex . Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells . S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway . In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23 . The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein . The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock . The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex. J Biol Chem, 2004 Feb 13, 279(7), 5353 - 62 Epub 2003 Nov 24. Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators; Chapman-Smith A et al.; The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR) . The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain . Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes . Here, we have analyzed purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1alpha and evaluated the contribution of the PAS domains to DNA binding in vitro . Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1alpha with the corresponding region of Arnt . Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences required the PAS A domains and exhibited KD values of 0.4 nM and approximately 50 nM, respectively . In contrast, the presence of the PAS domains of Arnt had little effect on DNA binding by Arnt homodimers, and these bound DNA with a KD of 45 nM . In the case of the DR, both high affinity DNA binding and dimer stability were specific to its native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generated a destabilized protein that bound DNA poorly. J Biol Chem, 2004 Feb 13, 279(7), 5861 - 6 Epub 2003 Nov 24. Amino acid substitution at the dimeric interface of human manganese superoxide dismutase; Hearn AS et al.; The side chains of His30 and Tyr166 from adjacent subunits in the homotetramer human manganese superoxide dismutase (Mn-SOD) form a hydrogen bond across the dimer interface and participate in a hydrogen-bonded network that extends to the active site . Compared with wild-type Mn-SOD, the site-specific mutants H30N, Y166F, and the corresponding double mutant showed 10-fold decreases in steady-state constants for catalysis measured by pulse radiolysis . The observation of no additional effect upon the second mutation is an example of cooperatively interacting residues . A similar effect was observed in the thermal stability of these enzymes; the double mutant did not reduce the major unfolding transition to an extent greater than either single mutant . The crystal structures of these site-specific mutants each have unique conformational changes, but each has lost the hydrogen bond across the dimer interface, which results in a decrease in catalysis . These same mutations caused an enhancement of the dissociation of the product-inhibited complex . That is, His30 and Tyr166 in wild-type Mn-SOD act to prolong the lifetime of the inhibited complex . This would have a selective advantage in blocking a cellular overproduction of toxic H2O2. J Biol Chem, 2004 Feb 20, 279(8), 6994 - 7000 Epub 2003 Nov 24. Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin; Lee JM et al.; In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum . Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca2+, but also to the active Ca2+ release process during excitation-contraction coupling . Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin . Using in vitro binding assay and site-directed mutagenesis, we found that the second intraluminal loop of the skeletal muscle RyR1 (amino acids 4860-4917), but not the first intraluminal loop of RyR1 (amino acids 4581-4640) could bind triadin . Specifically, three negatively charged residues Asp4878, Asp4907, and Glu4908 appear to be critical for the association with triadin . Using deletional approaches, we showed that a KEKE motif of triadin (amino acids 200-232) is essential for the binding to RyR1 . Because the second intraluminal loop of RyR has been previously shown to contain the ion-conducting pore as well as the selectivity filter of the Ca2+ release channel, and Asp4878, Asp4907, and Glu4908 residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca2+ release process of excitation-contraction coupling in muscle cells. Glycobiology, 2004 Mar, 14(3), 253 - 63 Epub 2003 Nov 24. Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants; Newburg DS et al.; To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens . Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs . Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked . The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides . Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001) . Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios . The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 {SE}) than the remaining infants (8.5 +/- 0.8; p<0.001) . Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042) . Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea . We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk. Antimicrob Agents Chemother, 2003 Dec, 47(12), 3704 - 7 Effect of moxifloxacin on production of proinflammatory cytokines from human peripheral blood mononuclear cells; Choi JH et al.; The effects of moxifloxacin, a new methoxyfluoroquinolone, on the production of proinflammatory cytokines from human peripheral blood mononuclear cells (PBMCs) were evaluated . Moxifloxacin inhibited