Escherichia coli

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 the production of tumor necrosis factor alpha (TNF-alpha) and/or interleukin-6 (IL-6) by PBMCs stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), and heat-killed bacteria in a concentration-dependent manner without cytotoxic effects . The addition of moxifloxacin reduced the population of cells positive for CD-14 and TNF-alpha and for CD-14 and IL-6 among the LPS- or LTA-stimulated PBMCs . By Western blot analysis, moxifloxacin pretreatment reduced the degradation of IkappaBalpha in LPS-stimulated PBMCs . In conclusion, moxifloxacin could interfere with NF-kappaB activation by inhibiting the degradation of IkappaBalpha and reduce the levels of production of proinflammatory cytokines.

Mutat Res, 2003 Oct 29, 531(1-2), 205 - 17
Chemical rearrangement and repair pathways of 1,N6-ethenoadenine; Speina E et al.; 1,N(6)-Ethenoadenine (epsilonA) is an exocyclic DNA adduct introduced to DNA by vinyl chloride and related compounds as well as in the consequence of oxidative stress and lipid peroxidation (LPO) . This highly genotoxic DNA damage is chemically unstable and either depurinates or converts into pyrimidine ring-opened secondary lesions . We have studied the structures of derivatives formed during epsilonA chemical rearrangement and identified enzymes repairing one of the rearrangement products . Rearrangement involves a water molecule addition to the C(2)-N(3) bond of epsilonA, resulting in formation of pyrimidine ring-closed B1 product, which is in equilibrium with pyrimidine ring-opened B2 compound . B2 further deformylates to yield compound C . N-Glycosidic bond of compound C is unstable and C depurinates, yielding compound D . These secondary lesions are not repaired by alkylpurine DNA N-glycosylase, which excises the parental epsilon A . Compound B, when paired with thymine and cytosine is efficiently excised by Escherichia coli formamidopirymidine DNA N-glycosylase (Fpg), and thymine glycol DNA N-glycosylases from E . coli (Nth) and Saccharomyces cerevisiae (Ntg2) . B is eliminated from B:G pair only by Nth and Ntg2 glycosylases, however none of the enzymes studied is excising B from B:A pair . This enables finishing of rearrangement, formation of AP sites and subsequently DNA strand breaks . During in vitro translesion synthesis, C is much easier bypassed by DNA polymerases, than compound B, and also than the parental epsilonA as well as than the AP site . This bypass beyond C proceeds mainly by misinsertion of adenine and guanine, or by insertion of thymine, the latter restoring the parental A:T pair . Alternatively, looping out of adducted nucleotide alone or with adjacent one generates one- or two-nucleotide deletions . This may explain the previously reported 20-fold higher mutagenic potency of product C in comparison to epsilon A in E . coli {Biochemistry 32 (1993) 12793}.

Mutat Res, 2003 Oct 29, 531(1-2), 141 - 56
Recognition of damaged DNA by Escherichia coli Fpg protein: insights from structural and kinetic data; Zharkov DO et al.; Formamidopyrimidine-DNA glycosylase (Fpg) excises oxidized purines from damaged DNA . The recent determination of the three-dimensional structure of the covalent complex of DNA with Escherichia coli Fpg, obtained by reducing the Schiff base intermediate formed during the reaction {Gilboa et al., J . Biol . Chem . 277 (2002) 19811} has revealed a number of potential specific and non-specific interactions between Fpg and DNA . We analyze the structural data for Fpg in the light of the kinetic and thermodynamic data obtained by the method of stepwise increase in ligand complexity to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for this enzyme {Ishchenko et al., Biochemistry 41 (2002) 7540} . Stopped-flow kinetic analysis that has allowed the dissection of Fpg catalysis in time {Fedorova et al., Biochemistry 41 (2002) 1520} is also correlated with the structural data.

Biochem Pharmacol, 2003 Dec 15, 66(12), 2333 - 40
Effects of cytochrome b(5) on drug oxidation activities of human cytochrome P450 (CYP) 3As: similarity of CYP3A5 with CYP3A4 but not CYP3A7; Yamaori S et al.; Effects of cytochrome b(5) (b(5)) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates . The activities of CYP3A5 were enhanced by addition of b(5) in approximately one third of the substrates employed in this study . Such enhancement by b(5) was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b(5) with any substrates employed . V(max) values for midazolam 1'-hydroxylation and amitriptyline N-demethylation by CYP3A5 were increased about twice by addition of b(5), which was also seen with CYP3A4, although the extent of the effects of b(5) on S(50) (K(m)) and Hill coefficient differed dependent on substrates used . In contrast, b(5) did not alter any of these kinetic parameters of CYP3A7 . The effects of b(5) on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7 . These results suggest that roles of b(5) in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7.

Vet Microbiol, 2003 Dec 2, 97(1-2), 87 - 101
Virulence genes of O149 enterotoxigenic Escherichia coli from outbreaks of postweaning diarrhea in pigs; Noamani BN et al.; The goal of this research was to determine whether isolates of O149 porcine enterotoxigenic Escherichia coli (ETEC) recovered from recent outbreaks of severe diarrhea in weaned pigs in Ontario, Canada, had virulence attributes different from those of isolates of the same serogroup from diarrhea of pigs in the 1970s and 1980s . Polymerase chain reaction amplification was used to determine the distribution of 11 virulence-associated genes in recent (100 isolates) and old (35 isolates) Ontario O149 porcine ETEC . These tests demonstrated that 92% of the recent isolates possessed the estA gene for STa enterotoxin, whereas none of the old isolates had this gene . H antigen determination showed that all the isolates which lacked the estA gene (all 35 old isolates plus 8 recent isolates) were H43, whereas isolates which had the estA gene were H10 . The astA gene for enteroaggregative heat-stable enterotoxin (EAST1) and the K88ac antigen were present in all 135 isolates . Plasmid analyses identified a cryptic 5.1kb plasmid in 99% of recent and 60% of old isolates . Suppressive subtractive hybridization associated several types of DNA fragments with the recent O149 ETEC, namely, fragments with no homology to DNA in databases, fragments of LPS biosynthesis genes, and F plasmid DNA . We conclude that the recent outbreaks of PWD in Ontario pigs were associated primarily with a new serotype of O149 ETEC and that isolates of this serotype possessed the estA gene that was not present in old O149 ETEC isolated from pigs in Ontario.

Gene, 2003 Dec 4, 321, 163 - 71
Blocking the T4 lysis inhibition phenotype; Slavcev RA et al.; Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4rII phage termed lysis inhibition (LIN) . E . coli K cells expressing lambda rexB from either a prophage defective for rexA, or a multicopy plasmid supported T4rII infection, but prevented the establishment of LIN . In addition, E . coli null mutations in either the periplasmic "tail-specific protease" tsp, or the 10Sa RNA ssrA, completely blocked the establishment of LIN following T4 infections . The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of lambda S and T4t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures . These activities of RexB were impeded in the presence of RexA.

Biochim Biophys Acta, 2003 Nov 20, 1639(3), 159 - 68
Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro; Ksiezak-Reding H et al.; Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling . The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt . We studied Akt-dependent phosphorylation of tau protein in vitro . We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau . Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1 . The Akt-phosphorylated tau retained its initial electrophoretic mobility . Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100 . Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3beta activity . On the other hand, GSK3beta selectively blocked Ser214 phosphorylation, which was prevented by lithium . The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases . Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.

Mol Cell, 2003 Nov, 12(5), 1309 - 15
Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2'-O-ribose methylation of a conserved rRNA position in yeast; Bonnerot C et al.; In yeast, guide snoRNAs have been assigned to 51 of the 55 rRNA ribose methylation sites . LSU-Um2918 is one of the four remaining positions . This residue is highly conserved and located in the peptidyl transferase center of the ribosome . The equivalent position on the E . coli 23S rRNA is methylated by FtsJ/RrmJ which has three yeast homologs: Spb1, involved in biogenesis of LSU; Trm7, a tRNA methyltransferase; and Mrm2, a mitochondrial 21S rRNA methyltransferase . We demonstrate that a point mutation in the Ado-Met binding site of Spb1p affects cell growth but does not abolish methylation of U2918 . When this mutation is combined with disruption of snR52 (a snoRNA C/D), cell growth is severely impaired and U2918 is no longer methylated . In vitro, Spb1p is able to methylate U2918 on 60S subunits . Our results reveal the importance of this methylation for which two mechanisms coexist: a site-specific methyltransferase (Spb1p) and a snoRNA-dependent mechanism.

Mol Cell, 2003 Nov, 12(5), 1077 - 86
Mechanism of 5'-directed excision in human mismatch repair; Genschel J et al.; We have developed a purified system that supports mismatch-dependent 5'-->3' excision . In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair . MutSalpha confers high processivity on EXOI, and termination is due to RPA-dependent displacement of this processive complex from the helix and a weak ability of EXOI to reload at the RPA-bound gap in the product, as well as MutSalpha- and MutLalpha-dependent suppression of EXOI activity in the absence of a mismatch cofactor . As observed in the purified system, excision directed by a 5' strand break in HeLa nuclear extract can proceed in the absence of MutLalpha or PCNA, although 3' excision in the extract system requires both proteins.

Scand J Immunol, 2003 Dec, 58(6), 613 - 9
Endotoxin tlerance inhibits lipopolysaccharide-initiated acute pulmonary inflammation and lung injury in rats by the mechanism of nuclear factor-kappaB; Qu J et al.; In this study, the effect of endotoxin tolerance on lipopolysaccharide (LPS)-initiated pulmonary inflammation, the local production of tumour necrosis factor-alpha (TNF-alpha) and the cytokine-induced neutrophil attractant (CINC), as well as the activation of nuclear factor-kappaB (NF-kappaB) and its subunit composition, were examined in vivo . Endotoxin tolerance was reproduced by four consecutive daily intraperitoneal injections of 0.6 mg/kg of Escherichia coli 055:B5 LPS . Compared with control rats, endotoxin-tolerant rats failed to increase the permeability of pulmonary microvascular or recruit neutrophil to lung tissue upon restimulation with 6 mg/kg of LPSs . Pretreatment with LPSs inhibited the protein level of TNF-alpha in bronchoalveolar lavage fluid (BALF) and mRNA expression of CINC in lung tissue in response to subsequent LPS stimulation . These changes were accompanied by the suppression of activation of NF-kappaB, including the low level of total amount of DNA-binding activity and high percentage of non-transactive p50 homodimers . These data demonstrate that endotoxin tolerance can alleviate the LPS-induced acute neutrophilic pulmonary inflammation in rats and can inhibit the proinflammatory cytokines in lung and suggest that endotoxin tolerance might result from the unresponsiveness of NF-kappaB and persistent high percentage of p50 homodimers . Therefore, the phenomenon of endotoxin tolerance might be used as a strategy for the prevention or treatment of LPS-associated acute respiratory distress syndrome in which excessive or dysregulated inflammation leads to acute lung injury.

Biochemistry, 2003 Dec 2, 42(47), 14047 - 56
Core formation in Escherichia coli bacterioferritin requires a functional ferroxidase center; Baaghil S et al.; Bacterioferritin from Escherichia coli is able to accumulate large quantities of iron in the form of an inorganic iron(III) mineral core . Core formation in the wild-type protein and a number of ferroxidase center variants was studied to determine key features of the core formation process and, in particular, the role played by the ferroxidase center . Core formation rates were found to be iron(II)-dependent and also depended on the amount of iron already present in the core, indicating the importance of the core surface in the mineralization reaction . Core formation was also found to be pH-dependent in terms of both rate and iron-loading characteristics, occurring with maximum efficiency at pH 6.5 . Even at this optimum pH, however, the effective iron capacity was approximately 2700 per molecule, i.e., well below the theoretical limit of approximately 4500, suggesting that competing oxidation/precipitation processes have a major influence on the amount of iron accumulated . Disruption of the ferroxidase center, by site-directed mutagenesis or by chemical inhibition with zinc(II), had a profound effect on core formation . Effective iron capacities were found to be linked to iron(II) oxidation rates, and in zinc(II)-inhibited wild-type and E18A bacterioferritins core formation was severely restricted . Zinc(II) was also able, even at low stoichiometries (12-60 ions/protein), to significantly inhibit further core formation in protein already containing a substantial core, indicating the importance of the ferroxidase center throughout the core formation process . A mechanism is proposed that incorporates essential roles for the core surface and the ferroxidase center . A central feature of this mechanism is that dioxygen cannot readily gain access to the core, perhaps because the channels through the bacterioferritin coat are hydrophilic and dioxygen is nonpolar.

Biochemistry, 2003 Dec 2, 42(47), 14004 - 16
Perturbation from a distance: mutations that alter LacI function through long-range effects; Swint-Kruse L et al.; Allosteric modification of ligand binding is central to LacI transcription control . Recently, the conformational change between LacI operator- and inducer-bound states was simulated with targeted molecular dynamics (TMD) {Flynn, T . C., Swint-Kruse, L., Kong, Y., Booth, C., Matthews, K . S., and Ma, J . (2003) Protein Sci., 12, 2523-2541} . Atomic-level analyses of TMD results indicate the structural importance of the core pivot region that connects the N- and C-subdomains flanking the inducer-binding site . Further, a number of LacI mutations in the core pivot have been identified recently by their altered behaviors in phenotypic screens . Biochemical characterization of three of these variants-L148F, S151P, and P320A-provides an opportunity to directly explore the role of the core pivot in repressor function . For L148F, inducer IPTG binding affinity is strengthened, whereas O(1) operator DNA binding is diminished approximately 30-fold . In contrast, O(1) binding is increased for S151P, whereas IPTG binding is decreased . UV-difference spectroscopy and urea denaturation indicate long-range effects in both variants . Interestingly, P320A binds to DNA approximately 4-fold more tightly than wild-type, yet inducer binding is unaffected . To examine linkage between the core pivot and DNA binding domains, the L148F substitution was combined with Q60G, a previously known mutant with enhanced operator affinity . The double mutant exhibits the properties of both parent proteins, resulting in near wild-type DNA binding affinity and enhanced inducer sensitivity . These features may render Q60G/L148F more cost-effective in technological applications than wild-type repressor . As a group, the behaviors of the core pivot mutants are consistent with the allosteric structural role predicted for this region by TMD and reflect the significant long-range impact that single substitutions can elicit on protein function.

Biochemistry, 2003 Dec 2, 42(47), 13887 - 92
Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro; Trivedi VD et al.; A chimeric fusion protein consisting of Natronomonas pharaonis sensory rhodopsin II (SRII), fused by a flexible linker to the two transmembrane helices of its cognate transducer protein, HtrII, followed by the HtrII membrane-proximal cytoplasmic fragment joined to the cytoplasmic domains of the Escherichia coli chemotaxis receptor Tsr, was expressed in E . coli . Purified fusion chimera protein reconstituted in liposomes binds to E . coli CheA kinase in the presence of the coupling protein CheW, and activates CheA autophosphorylation activity . CheA kinase activity is stimulated by photoexcitation of the SRII domain of the fusion protein, as shown by the wavelength-dependence of photostimulated phosphotransfer to the E . coli flagellar motor response regulator CheY in the purified in vitro liposomal system . Further confirming the fidelity of the in vitro system, increased and decreased levels of CheA activation in vitro result from overmethylated and undermethylated fusion protein purified from methylesterase and methyltransferase-deficient E . coli, respectively . Photoexcitation of the undermethylated fusion protein resulted in a 3-fold increase in phosphotransfer over that of the dark state . The results directly demonstrate the coupling of SRII photoactivated states to histidine kinase activity, previously predicted on the basis of sequence homologies of the haloarchaeal phototaxis system components to those of E . coli chemotaxis . The fusion chimera provides the first tool for in vitro measurement of photosignaling activity of SRII-HtrII molecular complexes.

Biochemistry, 2003 Dec 2, 42(47), 13848 - 55
Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain; Callaghan AJ et al.; RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria . Most endoribonucleases have been shown to act distributively; however, Feng et al . {(2002) Proc . Natl . Acad . Sci . U.S.A . 99, 14746-14751} have recently found that RNase E acts via a scanning mechanism . A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E . coli RNase E forms a homotetramer . Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue . The tetrameric assembly of the N-terminal domain of RNase E is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D(2) dihedral symmetry . Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer . This shape, together with limited proteolysis data, suggests that the S1-RNA binding domains of RNase E lie on the periphery of the tetramer . These observations have implications for the structure and function of the RNase E/RNase G ribonuclease family and for the assembly of the E . coli RNA degradosome, in which RNase E is the central component.

J Comput Aided Mol Des, 2003 May-Jun, 17(5-6), 383 - 97
Binding of alpha-hydroxy-beta-amino acid inhibitors to methionine aminopeptidase . The performance of two types of scoring functions; Jorgensen AT et al.; The binding mode of a recently described set of alpha-hydroxy-beta-amino acid inhibitors of methionine aminopeptidase type 2 is suggested in the present work . The binding mode is supported by analysis of published structures of transition state analogues co-crystallised with E . coli methionine aminopeptidase and by a comparison of molecular interaction fields calculated using GRID for E . coli and human methionine aminopeptidase . Based on the suggested binding mode two types of scoring functions have been evaluated and compared . These are the commercially available consensus score, CScore, and scoring of the ligands employing energies calculated using the Merck Molecular Force Field (MMFF) . Enriched subsets of ligands were obtained when using CScore, but these scores could not be used to assess the relative affinities of individual compounds . Although still not sufficiently accurate for reliable predictive purposes, an improved correlation was obtained between structure and affinity using a combined force field energy including contributions from solvation and conformational energy penalty for binding.

J Comput Aided Mol Des, 2003 May-Jun, 17(5-6), 367 - 82
Molecular model of the neural dopamine transporter; Ravna AW et al.; The dopamine transporter (DAT) regulates the action of dopamine by reuptake of the neurotransmitter into presynaptic neurons, and is the main molecular target of amphetamines and cocaine . DAT and the Na+/H+ antiporter (NhaA) are secondary transporter proteins that carry small molecules across a cell membrane against a concentration gradient, using ion gradients as energy source . A 3-dimensional projection map of the E . coli NhaA has confirmed a topology of 12 membrane spanning domains, and was previously used to construct a 3-dimensional NhaA model with 12 trans-membrane alpha-helices (TMHs) . The NhaA model, and site directed mutagenesis data on DAT, were used to construct a detailed 3-dimensional DAT model using interactive molecular graphics and empiric force field calculations . The model proposes a dopamine transport mechanism involving TMHs 1, 3, 4, 5, 7 and 11 . Asp79, Tyr252 and Tyr274 were the primary cocaine binding residues . Binding of cocaine or its analogue, (-)-2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT), seemed to lock the transporter in an inactive state, and thus inhibit dopamine transport . The present model may be used to design further experimental studies of the molecular structure and mechanisms of DAT and other secondary transporter proteins.

Bioessays, 2003 Dec, 25(12), 1168 - 77
Macromolecular complexes that unwind nucleic acids; von Hippel PH et al.; In this essay, we consider helicases, defined as enzymes that use the free energies of binding and hydrolysis of ATP to drive the unwinding of double-stranded nucleic acids, and ask how they function within, and are "coupled" to, the macromolecular machines of gene expression . To illustrate the principles of the integration of helicases into such machines, we consider the macromolecular complexes that direct and control DNA replication and DNA-dependent RNA transcription, and use these systems to illustrate how machines centered around coupled polymerase-helicase systems can be regulated by small changes in the interactions of their functional components .

Proteins, 2003 Dec 1, 53(4), 806 - 16
Prediction of deleterious functional effects of amino acid mutations using a library of structure-based function descriptors; Herrgard S et al.; An automated, active site-focused, computational method is described for use in predicting the effects of engineered amino acid mutations on enzyme catalytic activity . The method uses structure-based function descriptors (Fuzzy Functional Forms trade mark or FFFs trade mark ) to automatically identify enzyme functional sites in proteins . Three-dimensional sequence profiles are created from the surrounding active site structure . The computationally derived active site profile is used to analyze the effect of each amino acid change by defining three key features: proximity of the change to the active site, degree of amino acid conservation at the position in related proteins, and compatibility of the change with residues observed at that position in similar proteins . The features were analyzed using a data set of individual amino acid mutations occurring at 128 residue positions in 14 different enzymes . The results show that changes at key active site residues and at highly conserved positions are likely to have deleterious effects on the catalytic activity, and that non-conservative mutations at highly conserved residues are even more likely to be deleterious . Interestingly, the study revealed that amino acid substitutions at residues in close contact with the key active site residues are not more likely to have deleterious effects than mutations more distant from the active site . Utilization of the FFF-derived structural information yields a prediction method that is accurate in 79-83% of the test cases . The success of this method across all six EC classes suggests that it can be used generally to predict the effects of mutations and nsSNPs for enzymes . Future applications of the approach include automated, large-scale identification of deleterious nsSNPs in clinical populations and in large sets of disease-associated nsSNPs, and identification of deleterious nsSNPs in drug targets and drug metabolizing enzymes .

Proteins, 2003 Dec 1, 53(4), 777 - 82
Conserved structural elements in glutathione transferase homologues encoded in the genome of Escherichia coli; Rife CL et al.; Multiple sequence alignments of the eight glutathione (GSH) transferase homologues encoded in the genome of Escherichia coli were used to define a consensus sequence for the proteins . The consensus sequence was analyzed in the context of the three-dimensional structure of the gst gene product (EGST) obtained from two different crystal forms of the enzyme . The enzyme consists of two domains . The N-terminal region (domain I) has a thioredoxin-like alpha/beta-fold, while the C-terminal domain (domain II) is all alpha-helical . The majority of the consensus residues (12/17) reside in the N-terminal domain . Fifteen of the 17 residues are involved in hydrophobic core interactions, turns, or electrostatic interactions between the two domains . The results suggest that all of the homologues retain a well-defined group of structural elements both in and between the N-terminal alpha/beta domain and the C-terminal domain . The conservation of two key residues for the recognition motif for the gamma-glutamyl-portion of GSH indicates that the homologues may interact with GSH or GSH analogues such as glutathionylspermidine or alpha-amino acids . The genome context of two of the homologues forms the basis for a hypothesis that the b2989 and yibF gene products are involved in glutathionylspermidine and selenium biochemistry, respectively .

Arch Microbiol, 2004 Jan, 181(1), 26 - 34 Epub 2003 Nov 21.
Dependency of sugar transport and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system on membranous phosphatidylethanolamine in Escherichia coli: studies with a pssA mutant lacking phosphatidylserine synthase; Aboulwafa M et al.; An isogenic pair of Escherichia coli strains lacking ( pssA) and possessing (wild-type) the enzyme phosphatidylserine synthase was used to estimate the effects of the total lack of phosphatidylethanolamine (PE), the major phospholipid in E . coli membranes, on the activities of several sugar permeases (enzymes II) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . The mutant exhibits greatly elevated levels of phosphatidylglycerol (PG), a lipid that has been reported to stimulate the in vitro activities of several PTS permeases . The activities, thermal stabilities, and detergent sensitivities of three PTS permeases, the glucose enzyme II (II(Glc)), the mannose enzyme II (II(Man)) and the mannitol enzyme II (II(Mtl)), were characterized . Western blot analyses revealed that the protein levels of II(Glc) were not appreciably altered by the loss of PE . In the pssA mutant, II(Glc) and II(Man) activities were depressed both in vivo and in vitro, with the in vivo transport activities being depressed much more than the in vitro phosphorylation activities . II(Mtl) also exhibited depressed transport activity in vivo but showed normal phosphorylation activities in vitro . II(Man) and II(Glc) exhibited greater thermal lability in the pssA mutant membranes than in the wild-type membranes, but II(Mtl) showed enhanced thermal stability . All three enzymes were activated by exposure to TritonX100 (0.4%) or deoxycholate (0.2%) and inhibited by SDS (0.1%), but II(Mtl) was the least affected . II(Man) and, to a lesser degree, II(Glc) were more sensitive to detergent treatments in the pssA mutant membranes than in the wild-type membranes while II(Mtl) showed no differential effect . The results suggest that all three PTS permeases exhibit strong phospholipid dependencies for transport activity in vivo but much weaker and differential dependencies for phosphorylation activities in vitro, with II(Man) exhibiting the greatest and II(Mtl) the least dependency . The effects of lipid composition on thermal sensitivities and detergent activation responses paralleled the effects on in vitro phosphorylation activities . These results together with those previously published suggest that, while the in vivo transport activities of all PTS enzymes II require an appropriate anionic to zwitterionic phospholipid balance, the in vitro phosphorylation activities of these same enzymes show much weaker and differential dependencies . Alteration of the phospholipid composition of the membrane thus allows functional dissection of transport from the phosphorylation activities of PTS enzyme complexes.

Anal Bioanal Chem, 2004 Apr, 378(7), 1710 - 5 Epub 2003 Nov 22.
A rapid enzyme assay for beta-galactosidase using optically gated sample introduction on a microfabricated chip; Xu H et al.; The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction . The hydrolysis of fluorescein mono- beta- d-galactopyranoside (FMG) by beta- d-galactosidase ( beta-Gal) is continuously monitored using a microchip for 5 to 10 min . The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein . Kinetic information about beta-Gal has been successfully obtained by varying the concentration of FMG . beta-Gal enzymes from two different sources including bovine liver and E . coli., have been examined and compared to each other and to results obtained using traditional assay methods . In addition, the competitive inhibition of beta-Gal by phenylethyl beta- d-thiogalactoside (PETG) and beta-lactose has been studied using this technique . PETG is found to have higher inhibition than lactose in the hydrolysis . This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method . Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method . By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays . This technique should therefore be valuable for high-throughput screening in the drug discovery industry.

Nature, 2003 Dec 11, 426(6967), 684 - 7 Epub 2003 Nov 23.
Backtracking by single RNA polymerase molecules observed at near-base-pair resolution; Shaevitz JW et al.; Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo . Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events . The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3' end of the nascent RNA transcript away from the enzyme active site . The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site . Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP . Backtracking events ( approximately 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min . Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.

Am J Obstet Gynecol, 2003 Nov, 189(5), 1458 - 66
Betamethasone effects on chorioamnionitis induced by intra-amniotic endotoxin in sheep; Newnham JP et al.; OBJECTIVE: Intra-amniotic administration of endotoxin in sheep is a model of subclinical chorioamnionitis . Intrauterine inflammation alters lung development to improve postnatal lung function and may predispose the infant to lung and brain injury . We describe the effects of intra-amniotic endotoxin on cytokines and white cell responses in the membranes and amniotic fluid and investigate the hypothesis that betamethasone treatment suppresses these responses . STUDY DESIGN: Pregnant ewes were allocated at random to receive either intra-amniotic saline solution (control animals), maternal intramuscular betamethasone, intra-amniotic endotoxin by ultrasound guidance (10 mg Escherichia coli 055:B5), or a combination of the betamethasone and endotoxin treatments . Lambs were delivered abdominally at 110 to 125 days of gestation at time points that ranged from 2 hours to 15 days after treatment . RESULTS: When compared with saline solution-injected control animals, the intra-amniotic injection of endotoxin increased white cell counts in amniotic fluid . Levels of interleukin-8, but not interleukin-6, were significantly increased in amniotic fluid from 5 hours to 15 days after intra-amniotic endotoxin injection, and interleukin-8 levels were not decreased by concurrent treatment with betamethasone . After endotoxin treatment, interleukin-1beta and interleukin-8 messenger RNA were expressed in chorion, and interleukin-6 messenger RNA expression was localized to chorionic blood vessel epithelium . The half-life of endotoxin in the amniotic fluid was 1.7 days, and levels remained measurable 15 days after injection . CONCLUSION: These findings confirm that the fetus can survive within amniotic fluid that contains endotoxin, white cells, and cytokines for periods of weeks or more . Betamethasone treatment can suppress the initial inflammation in the amnion-chorion, but interleukin-8 levels and inflammatory cells in amniotic fluid were not suppressed 5 and 15 days after betamethasone treatment, presumably because of the slow clearance of bioactive endotoxin from the amniotic fluid.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14822 - 7 Epub 2003 Nov 21.
DNA bending and unbending by MutS govern mismatch recognition and specificity; Wang H et al.; DNA mismatch repair is central to the maintenance of genomic stability . It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins . Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair . To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy . Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites . These results suggest that the specific recognition complex is one in which the DNA is unbent . Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent . Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.

Plant Cell Physiol, 2003 Nov, 44(11), 1168 - 75
A new 9-lipoxygenase cDNA from developing rice seeds; Mizuno K et al.; We isolated a novel C9 position specific lipoxygenase (r9-LOX1) cDNA from developing rice seeds . The enzymatic features of r9-LOX1 resembled those of rice LOX-L3 known to be contained in rice germ and to have C9-specific LOX activity . However, the expression level of the r9-LOX1 gene was higher in imbibed seeds rather than developing seeds . A homology search against the rice nucleotide database revealed the r9-LOX1 gene to be on rice chromosome 3 (accession number AC093017) . The restriction enzyme map of the reported genomic sequence agreed with the result of the Southern blot analysis for the r9-LOX1 . The enzyme could be useful for in vitro synthesis of 9,10-ketol-octadecadienoic acid.

J Leukoc Biol, 2004 Feb, 75(2), 358 - 72 Epub 2003 Nov 21.
Gene expression in mature neutrophils: early responses to inflammatory stimuli; Zhang X et al.; Neutrophils provide an essential defense against bacterial and fungal infection and play a major role in tissue damage during inflammation . Using oligonucleotide microarrays, we have examined the time course of changes in gene expression induced by stimulation with live, opsonized Escherichia coli, soluble lipopolysaccharide, and the chemoattractant formyl-methionyl-leucyl-phenylalanine . The results indicate that activated neutrophils generate a broad and vigorous set of alterations in gene expression . The responses included changes in the levels of transcripts encoding 148 transcription factors and chromatin-remodeling genes and 95 regulators of protein synthesis or stability . Clustering analysis showed distinct temporal patterns with many rapid changes in gene expression within the first hour of exposure . In addition to the temporal clustering of genes, we also observed rather different profiles associated with each stimulus, suggesting that even a nonvirulent organism such as E . coli is able to play a dynamic role in shaping the inflammatory response . Principal component analysis of transcription factor genes demonstrated clear separation of the neutrophil-response clusters from those of resting and stimulated human monocytes . The present study indicates that combinatorial transcriptional regulation including alterations of chromatin structure may play a role in the rapid changes in gene expression that occur in these terminally differentiated cells.

J Pharmacol Exp Ther, 2004 Mar, 308(3), 887 - 95 Epub 2003 Nov 21.
Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes; Xu F et al.; Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure . Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid omega-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation . The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized . Substrate-binding assays indicated that leukotriene B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly . Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism . Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) of 9 and 11 min(-1), respectively . Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid omega-hydroxylase activity . The p450 omega-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed omega-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms . These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify p450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

J Biol Chem, 2004 Feb 20, 279(8), 6761 - 8 Epub 2003 Nov 20.
DsbB elicits a red-shift of bound ubiquinone during the catalysis of DsbA oxidation; Inaba K et al.; DsbB is an Escherichia coli plasma membrane protein that reoxidizes the Cys30-Pro-His-Cys33 active site of DsbA, the primary dithiol oxidant in the periplasm . Here we describe a novel activity of DsbB to induce an electronic transition of the bound ubiquinone molecule . This transition was characterized by a striking emergence of an absorbance peak at 500 nm giving rise to a visible pink color . The ubiquinone red-shift was observed stably for the DsbA(C33S)-DsbB complex as well as transiently by stopped flow rapid scanning spectroscopy during the reaction between wild-type DsbA and DsbB . Mutation and reconstitution experiments established that the unpaired Cys at position 44 of DsbB is primarily responsible for the chromogenic transition of ubiquinone, and this property correlates with the functional arrangement of amino acid residues in the neighborhood of Cys44 . We propose that the Cys44-induced anomaly in ubiquinone represents its activated state, which drives the DsbB-mediated electron transfer.

J Biol Chem, 2004 Feb 13, 279(7), 6027 - 34 Epub 2003 Nov 21.
Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators; Sharma S et al.; DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA . RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites . Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure . We constructed mutants in the two predicted DNA binding domains to test the model . As expected, the mutants were defective in P1 DNA binding . The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain . The experiments supported the prediction . The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member . These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.

EMBO J, 2003 Dec 1, 22(23), 6408 - 18
Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair; Lopez de Saro FJ et al.; Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins . In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family . We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved . Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself . Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site . Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.

EMBO J, 2003 Dec 1, 22(23), 6399 - 407
Decatenation of DNA circles by FtsK-dependent Xer site-specific recombination; Ip SC et al.; DNA replication results in interlinked (catenated) sister duplex molecules as a consequence of the intertwined helices that comprise duplex DNA . DNA topoisomerases play key roles in decatenation . We demonstrate a novel, efficient and directional decatenation process in vitro, which uses the combination of the Escherichia coli XerCD site-specific recombination system and a protein, FtsK, which facilitates simple synapsis of dif recombination sites during its translocation along DNA . We propose that the FtsK-XerCD recombination machinery, which converts chromosomal dimers to monomers, may also function in vivo in removing the final catenation links remaining upon completion of DNA replication.

EMBO J, 2003 Dec 1, 22(23), 6389 - 98
YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA; Kanehara K et al.; sigmaE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli . Under normal growth conditions, sigmaE activity is down-regulated by the membrane-bound anti-sigmaE protein, RseA . Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region . Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage) . Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent sigmaE activation . It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage . These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage.

EMBO J, 2003 Dec 1, 22(23), 6346 - 55
FinO is an RNA chaperone that facilitates sense-antisense RNA interactions; Arthur DC et al.; The protein FinO represses F-plasmid conjugative transfer by facilitating interactions between the mRNA of the major F-plasmid transcriptional activator, TraJ, and an antisense RNA, FinP . FinO is known to bind stem-loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense-antisense pairing is poorly understood . Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP . This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense-antisense pairing . The energy for FinO-catalyzed base-pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy . An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA-binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense-antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.

EMBO J, 2003 Dec 1, 22(23), 6193 - 204
Structural constraints on protein self-processing in L-aspartate-alpha-decarboxylase; Schmitzberger F et al.; Aspartate decarboxylase, which is translated as a pro-protein, undergoes intramolecular self-cleavage at Gly24-Ser25 . We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24-->Ser, His11-->Ala, Ser25-->Ala, Ser25-->Cys and Ser25-->Thr mutants . Comparative analyses of the cleavage site reveal specific conformational constraints that govern self-processing and demonstrate that considerable rearrangement must occur . We suggest that Thr57 Ogamma and a water molecule form an 'oxyanion hole' that likely stabilizes the proposed oxyoxazolidine intermediate . Thr57 and this water molecule are probable catalytic residues able to support acid-base catalysis . The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction . The molecular mechanism of self-processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate . Comparison of the structural features shows significant similarity to those in other self-processing systems, and suggests that models of the cleavage site of such enzymes based on Ser-->Ala or Ser-->Thr mutants alone may lead to erroneous interpretations of the mechanism.

Anesth Analg, 2003 Dec, 97(6), 1769 - 72
The dose-related effects of ketamine on mortality and cytokine responses to endotoxin-induced shock in rats; Taniguchi T et al.; In our previous study, ketamine administration was found to inhibit hypotension, metabolic acidosis, and cytokine responses in endotoxemia . However, only a few studies have indicated whether ketamine has the dose-related beneficial effects after endotoxin injection . Our objective was to clarify the dose-related effects of ketamine on mortality and cytokine responses to endotoxemia in rats . Sixty-five rats were divided at random among five equal groups: Group C was given saline alone . Group E was given endotoxin alone (Escherichia coli endotoxin; 10 mg/kg, IV) . Group L received a a low dose of ketamine (5 mg.kg(-1).h(-1), IV), Group M a medium dose of ketamine (10 mg.kg(-1).h(-1), IV), and Group H a high dose of ketamine (20 mg.kg(-1).h(-1), IV), all exposure to endotoxin . After endotoxin injection, hemodynamics, acid-base status, mortality rate, and plasma concentrations of tumor necrosis factor alpha and interleukin 6 were assessed for each of the five groups . Endotoxin injection produced progressive hypotension, metabolic acidosis, and a large increase in plasma cytokine concentrations . Mortality rates 8 h after endotoxin injection were 0% for group C, 92% for group E, 48% for group L, 0% for group M, and 32% for group H . Ketamine administration thus clearly had a beneficial effect on mortality rates, with that for group M lower than for groups L and H (P < 0.05) . The cytokine responses to endotoxin were somewhat suppressed in group M but not in group L . Ketamine administration dose-independently inhibited hypotension, metabolic acidosis, and cytokine responses in rats injected with endotoxin.

Anesth Analg, 2003 Dec, 97(6), 1756 - 63
The effects of vasopressin on systemic and splanchnic hemodynamics and metabolism in endotoxin shock; Martikainen TJ et al.; We compared the effects of vasopressin and norepinephrine on systemic and splanchnic circulation and metabolism in endotoxin shock in pigs . Twenty-one pigs were randomized to endotoxin shock (Escherichia coli endotoxin infusion) (n = 6), endotoxin and vasopressin (VASO; n = 6), endotoxin and norepinephrine (NE; n = 6), and controls (n = 3) . Endotoxin infusion was increased to induce hypotension, after which vasopressin or norepinephrine was started to keep systemic mean arterial blood pressure >70 mm Hg . Regional blood flows and arterial and regional lactate concentrations were measured . Tonometers with microdialysis capillaries were inserted into the stomach, jejunum, and colon . Systemic mean arterial blood pressure >70 mm Hg was achieved in the VASO and NE groups . Vasopressin decreased cardiac output, superior mesenteric artery, and portal vein blood flow, whereas hepatic arterial blood flow increased . Arterial lactate concentration increased from 2.0 mM (1.6-2.1 mM) to 4.7 mM (4.7-4.9 mM) (P = 0.007) . Systemic and mesenteric oxygen delivery and consumption decreased and oxygen extraction increased in the VASO group . Vasopressin increased mucosal-arterial PCO(2) gradients in all three locations, whereas luminal lactate release occurred only in the jejunum . Animals in the NE group remained stable . Vasopressin reversed hypotension but decreased systemic and gut blood flow . This was associated with hyperlactatemia, signs of visceral dysoxia, and jejunal luminal lactate release . IMPLICATIONS: Although vasopressin induces vasoconstriction in visceral region, its effects on splanchnic circulation and metabolism during septic-endotoxin shock are still poorly characterized . We evaluated the metabolic and hemodynamic effects of vasopressin and norepinephrine within the splanchnic area in porcine endotoxin shock.

J Vet Med B Infect Dis Vet Public Health, 2003 Oct, 50(8), 383 - 9
Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector; Hoelzle LE et al.; The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch . pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli . The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography . The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E . coli . Transformants expressing this full-length rMOMP were significantly reduced in viability . Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E . coli cytoplasm were used for immunization of rabbits . The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB . When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species . Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

Lett Appl Microbiol, 2003, 37(5), 380 - 5
Survival of nonspecific porin-deficient mutants of Escherichia coli in black sea water; Darcan C et al.; AIMS: The aim of this study was to investigate the links between survival of Escherichia coli in sea water microcosms in the laboratory and the presence of porins in the outer membrane . The E . coli strains studied were a wild-type strain and a series of outer membrane protein (omp) mutants . METHODS AND RESULTS: Bacteria were suspended in natural or filtered-autoclaved sea water microcosms and numbers determined over an incubation period by plate count and by count of cells capable of respiration . CONCLUSIONS: The type of omp mutation has a significant impact in bacterial survival . The double OmpC-OmpF mutant and the OmpR mutant (which was incapable of synthesizing OmpC and OmpF) survived poorly compared with single omp mutants and the wild-type strain . This suggests that these proteins are important in determining the entry of E . coli into the survival mode . The EnvZ mutant, which lacks the protein by which the cell senses some changes in the environment, survived as well as the wild-type strain when compared by plate counts and by respiring cell count . The loss of the EnvZ protein has no effect on survival but it could prevent the organism sensing the changes in the environment through which entry into the survival state is triggered . SIGNIFICANCE AND IMPACT OF THE STUDY: This work is another piece in the puzzle as to how bacteria survive stress conditions.

Lett Appl Microbiol, 2003, 37(6), 433 - 7
DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources; Domingue G et al.; AIMS: To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 . METHODS AND RESULTS: Eleven human and food strains isolated over a 15-year period were examined . All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant . Ten strains had VT2 genes which were all vtx2d . Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory . Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential . Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th . CONCLUSIONS: As a result of apparent clonality, PFGE proved essential for differentiation . Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences . SIGNIFICANCE AND IMPACT OF THE STUDY: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.

J Periodontal Res, 2003 Dec, 38(6), 591 - 6
Proteases augment the effects of lipopolysaccharide in rat gingiva; Ekuni D et al.; OBJECTIVES: Lipopolysaccharide (LPS) and proteases have been implicated as important factors in the initiation and progression of human periodontal diseases . A single application of LPS or proteases is insufficient to induce periodontal pocket formation or periodontitis . The aim of the present study was to assess the combined effect of lipopolysaccharide and proteases on rat periodontal tissues, and create a periodontal disease model . MATERIALS AND METHODS: Forty male Wistar rats were divided into four groups: combination group (treated with both LPS and proteases solutions); LPS group; proteases group; and control . Each solution was introduced daily into the palatal gingival sulcus of maxillary molars for 8 weeks . The tissues were evaluated histometrically and immunohistochemically . RESULTS: In the LPS group, elongation of rete ridge, apical migration of junctional epithelium (JE), increased numbers of B cells in connective tissue, and resorption of alveolar bone were observed . In the proteases group, the increase in the number of infiltrating polymorphonuclear leukocytes and blood vessels in the connective tissue was greater than that of the LPS group . CONCLUSIONS: The effects of LPS on periodontal tissues differed from those of proteases . The addition of proteases augmented and increased the effects of LPS, which were apical migration, intraepithelial cleavage of JE, and increased B cell density . The lesions in the combination group resembled established lesions of human periodontitis, with the exception of the low density of plasma cells.

J Antibiot (Tokyo), 2003 Sep, 56(9), 762 - 7
Molecular cloning of a D-cycloserine resistance gene from D-cycloserine-producing Streptomyces garyphalus; Matsuo H et al.; A 3.5-kb DNA fragment that confers resistance to D-cycloserine (DCS) was cloned from the chromosomal DNA of a DCS-producing Streptomyces garyphalus into Streptomyces lividans by a shot-gun cloning technique . Nucleotide sequence analysis revealed the existence of four open reading frames (ORFs B, C, D, and E), together with two incomplete ORFs, A and F . By introduction of the cloned fragment into Escherichia coli, the host obtained resistance to DCS . We showed that ORF B, which consists of 903 bp, is a DCS resistance gene . The hydropathy plot analysis of a protein deduced from ORF B revealed that the protein carries membrane-integral domains spanning the membrane 10 times, which suggests that the DCS-resistance determinant may be a factor associated with DCS transport.

Anal Chem, 2003 Aug 15, 75(16), 4113 - 9
A MutS-based protein chip for detection of DNA mutations; Bi LJ et al.; This paper describes a new protein chip method for detection of single-base mismatches and unpaired bases of DNA, using a genetic fusion molecular system Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutS (THLSLM) . The THLSLM coding sequence was constructed by attaching Strep-tag II and mutS gene to pET32a (+) sequentially with insertion of a linker peptide coding sequence before and behind Strep-tagII gene, respectively . THLSLM was expressed in E . coli AD494 (DE3) and purified using Ni(2+)-chelation affinity resin . THLSLM retained both mismatch recognition activity and streptavidin binding affinity . THLSLM was then immobilized on the chip matrix coated with streptavidin through the Strep-tag II-streptavidin binding reaction . The resulting protein chip was used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides, as well as a single-base mutation in rpoB gene from Mycobacterium tuberculosis, with high specificity . The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations.

Protein Eng, 2003 Nov, 16(11), 809 - 17
Change in the crystal packing of soybean beta-amylase mutants substituted at a few surface amino acid residues; Kang YN et al.; In spite of the high similarity of amino acid sequence and three-dimensional structure between soybean beta-amylase (SBA) and sweet potato beta-amylase (SPB), their quaternary structure is quite different, being a monomer in SBA and a tetramer in SPB . Because most of the differences in amino acid sequences are found in the surface region, we tested the tetramerization of SBA by examining mutations of residues located at the surface . We designed the SBA tetramer using the SPB tetramer structure as a model and calculating the change of accessible surface area (DeltaASA) for each residue in order to select sites for the mutation . Two different mutant genes encoding SB3 (D374Y/L481R/P487D) and SB4 (K462S added to SB3), were constructed for expression in Escherichia coli and the recombinant proteins were purified . They existed as a monomer in solution, but gave completely different crystals from the native SBA . The asymmetric unit of the mutants contains four molecules, while that of native SBA contains one . The interactions of the created interfaces revealed that there were more intermolecular interactions in the SB3 than in the SB4 tetramer . The substituted charged residues on the surface are involved in interactions with adjacent molecules in a different way, forming a new crystal packing pattern.

Protein Eng, 2003 Nov, 16(11), 795 - 8
The role of loop 7 in mediating calcineurin regulation; Xiang BQ et al.; Calcineurin (CN) is a heterodimer protein consisting of a 61 kDa catalytic subunit A and a 19 kDa regulatory subunit B . It plays a critical role in T-cell activation and is involved in many cellular processes . Regulation of CN is rather complex, including a number of factors such as divalent metal ions (primarily Ca(2+) and Mn(2+)), calmodulin (CaM) and autoinhibition (AI) segment . Previously, we reported that a loop 7 deletion mutant (V314) in subunit A exhibited high phosphatase activity, although the mechanism for the surprising activity enhancement and whether the activity change applies to other loop 7 residues were not known . In order to probe the role of loop 7, we have carried out extensive mutagenesis experiments, followed by systematic activity assays under a number of regulatory conditions . All mutants, including single deletion mutants Y315, N316 and double deletion mutant V314Y315, showed increased phosphatase activity . Significantly, activities of the mutants containing the V314 deletion, namely V314 and V314Y315, were no longer regulated by regulatory subunit B . These results, along with the structure analysis, suggest that loop 7 as a whole plays an important role in mediating CN's regulation through bridging the regulatory subunit and catalytic core and interaction with the AI segment of CN.

Plant Cell, 2003 Dec, 15(12), 3007 - 19 Epub 2003 Nov 20.
Engineering vitamin E content: from Arabidopsis mutant to soy oil; Van Eenennaam AL et al.; We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase . This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed . Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase . Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2% . These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition . When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to >95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity . These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14689 - 94 Epub 2003 Nov 20.
A peptide switch regulates DNA polymerase processivity; Lopez de Saro FJ et al.; Chromosomal DNA polymerases are tethered to DNA by a circular sliding clamp for high processivity . However, lagging strand synthesis requires the polymerase to rapidly dissociate on finishing each Okazaki fragment . The Escherichia coli replicase contains a subunit (tau) that promotes separation of polymerase from its clamp on finishing DNA segments . This report reveals the mechanism of this process . We find that tau binds the C-terminal residues of the DNA polymerase . Surprisingly, this same C-terminal "tail" of the polymerase interacts with the beta clamp, and tau competes with beta for this sequence . Moreover, tau acts as a DNA sensor . On binding primed DNA, tau releases the polymerase tail, allowing polymerase to bind beta for processive synthesis . But on sensing the DNA is complete (duplex), tau sequesters the polymerase tail from beta, disengaging polymerase from DNA . Therefore, DNA sensing by tau switches the polymerase peptide tail on and off the clamp and coordinates the dynamic turnover of polymerase during lagging strand synthesis.

Genes Dev, 2003 Nov 15, 17(22), 2839 - 51
Tethering sigma70 to RNA polymerase reveals high in vivo activity of sigma factors and sigma70-dependent pausing at promoter-distal locations; Mooney RA et al.; Bacterial sigma factors compete for binding to RNA polymerase (RNAP) to control promoter selection, and in some cases interact with RNAP to regulate at least the early stages of transcript elongation . However, the effective concentration of sigmas in vivo, and the extent to which sigma can regulate transcript elongation generally, are unknown . We report that tethering sigma70 to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding sigma70 and RNAP's beta' subunit, respectively) yields viable Escherichia coli strains in which alternative sigma-factor function is not impaired . beta'::sigma70 RNAP transcribed DNA normally in vitro, but allowed sigma70-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit . Based on measurement of the effective concentration of tethered sigma70, we conclude that the effective concentration of sigma70 in E . coli (i.e., its thermodynamic activity) is close to its bulk concentration . At this level, sigma70 would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.

Bioinformatics, 2003 Nov 22, 19(17), 2225 - 36
In silico analysis reveals substantial variability in the gene contents of the gamma proteobacteria LexA-regulon; Erill I et al.; MOTIVATION: Motif-prediction algorithm capabilities for the analysis of bacterial regulatory networks and the prediction of new regulatory sites can be greatly enhanced by the use of comparative genomics approaches . In this study, we make use of a consensus-building algorithm and comparative genomics to conduct an in-depth analysis of the LexA-regulon of gamma proteobacteria, and we use the inferred results to study the evolution of this regulatory network and to examine the usefulness of the control sequences and gene contents of regulons in phylogenetic analysis . RESULTS: We show, for the first time, the substantial heterogeneity that the LexA-regulon of gamma proteobacteria displays in terms of gene content and we analyze possible branching points in its evolution . We also demonstrate the feasibility of using regulon-related information to derive sound phylogenetic inferences . AVAILABILITY: Complementary analysis data and both the source code and the Windows-executable files of the consensus-building software are available at http://www.cnm.es/~ivan/RCGScanner/

Am J Physiol Gastrointest Liver Physiol, 2004 Apr, 286(4), G645 - 52 Epub 2003 Nov 20.
Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation; Kojima K et al.; Protection of colonic epithelial integrity and function is critical, because compromises in mucosal functions can lead to adverse and potentially life-threatening effects . The gut flora may contribute to this protection, in part, through the sustained induction of cytoprotective heat shock proteins (HSPs) in surface colonocytes . In this study, we investigated whether Escherichia coli LPS mediates bacteria-induced HSP by using cultured young adult mouse colon (YAMC) cells, an in vitro model of the colonic epithelium . E . coli LPS led to an epithelial cell-type specific induction of HSP25 in a time- and concentration-dependent manner, an effect that did not involve changes in HSP72 . YAMC cells expressed the toll-like receptors (TLR)2 and TLR4 but not the costimulatory CD14 molecule . Whereas LPS stimulated both the p38 and ERK1/2 but not the stress-activated protein kinase/c-Jun NH(2)-terminal kinase, signaling pathways in the YAMC cells, all three were stimulated in RAW macrophage cells (in which no LPS-induced HSP25 expression was observed) . The p38 inhibitor SB-203580 and the MAP kinase kinase-1 inhibitor PD-98059 inhibited HSP25 induction by LPS . LPS treatment also conferred protection against actin depolymerization induced by the oxidant monochloramine . The HSP25 dependence of the LPS protective effect was outlined in inhibitor studies and through adenovirus-mediated overexpression of HSP25 . In conclusion, LPS may be an important mediator of enteric bacteria-induced expression of intestinal epithelial HSP25, an effect that may contribute to filamentous actin stabilization under physiological as well as pathophysiological conditions and thus protection of colonic epithelial integrity.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Dec 5, 798(1), 145 - 54
Validated method for the simultaneous determination of Delta 9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC in human plasma using solid phase extraction and gas chromatography-mass spectrometry with positive chemical ionization; Gustafson RA et al.; A fully validated, highly sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THCCOOH) in plasma is presented . This method incorporates Escherichia coli beta-glucuronidase hydrolysis to cleave glucuronic acid moieties to capture total analyte concentrations, and simultaneous solid phase extraction (SPE) of the three analytes in a single eluant with separation and quantification on a bench-top positive chemical ionization (PCI) gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode . Quantitation was achieved by the addition of deuterated analogues for each analyte as internal standards (IS) . Limits of quantitation (LOQ) were 0.5, 0.5 and 1.0 for THC, 11-OH-THC and THCCOOH, respectively, with linearity ranging up to 50 ng/ml for THC and 11-OH-THC, and 100 ng/ml for THCCOOH . Absolute recoveries ranged from 67.3 to 83.5% for all three analytes . Intra-assay accuracy and precision ranged from 1.2 to 12.2 and 1.4 to 4.7%, respectively . Inter-assay accuracy and precision ranged from 1.4 to 12.2 and 3.1 to 7.3%, respectively . This method was used to analyze plasma samples collected from individuals participating in a controlled oral THC administration study . Statistically significant (P< or =0.05) increases of 40% for 11-OH-THC and 42% for THCCOOH concentrations were found between hydrolyzed and non-hydrolyzed results . This method will be utilized in ongoing controlled cannabinoid administration studies and may be a useful analytical procedure for the fields of forensic toxicology and cannabinoid pharmacology.

Neurosci Res, 2003 Dec, 47(4), 383 - 90
Ganglioside GM1 binding toxins and human neuropathy-associated IgM antibodies differentially promote neuritogenesis in a PC12 assay; O'Hanlon GM et al.; PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB) . Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies . Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay . Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic . Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA . The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic . Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects . These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA . Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.

FEBS Lett, 2003 Nov 27, 555(1), 160 - 9
Membrane protein reconstitution and crystallization by controlled dilution; Remigy HW et al.; Efficient reconstitution of membrane proteins for functional analyses can be achieved by dilution of a ternary mixture containing proteins, lipids and detergents . Once the dilution reaches the point where the free detergent concentration would become lower than the critical micellar concentration, detergent is recruited from the bound detergent pool, and association of proteins and lipids is initiated . Here we show that dilution is also suitable for the assembly of two-dimensional crystals . A device has been designed that allows controlled dilution of a protein-lipid-detergent mixture to induce formation of densely packed or crystalline proteoliposomes . Turbidity is used to monitor the progress of reconstitution on-line, while dilution is achieved by computer-controlled addition of buffer solution in sub-microliter steps . This system has mainly been tested with porin OmpF, a typical beta-barrel protein, and aquaporin-1, a typical alpha-helical protein . The results demonstrate that large, highly ordered two-dimensional crystals can be produced by the dilution method.

FEBS Lett, 2003 Nov 27, 555(1), 144 - 50
NMR solution structure determination of membrane proteins reconstituted in detergent micelles; Fernandez C et al.; As an alternative to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy in solution can be used for three-dimensional structure determination of small membrane proteins, preferably proteins with beta-barrel fold . This paper reviews recent achievements as well as limiting factors encountered in solution NMR studies of membrane proteins . Our particular interest has been focused on supplementing structure determination with data on the solvation of the proteins in the mixed micelles with detergents that are used to reconstitute membrane proteins for the NMR experiments . For the Escherichia coli outer membrane protein X (OmpX) in dihexanoylphosphatidylcholine (DHPC) micelles, such studies showed that the central part of the protein is covered with a fluid monolayer of lipid molecules, which seems to mimic quite faithfully the embedding of the protein in the lipid phase of the biological membrane . The implication is that the micellar systems used in this instance for the NMR studies of the membrane protein should also be suitable for further investigations of functional interactions with other proteins or low-molecular weight ligands.

FEBS Lett, 2003 Nov 27, 555(1), 122 - 5
Membrane protein folding: beyond the two stage model; Engelman DM et al.; The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization . Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process . Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.

FEBS Lett, 2003 Nov 27, 555(1), 111 - 5
The structures of BtuCD and MscS and their implications for transporter and channel function; Bass RB et al.; The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters . Although these transport families encompass a wide range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane . This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch-activated mechanosensitive channel of small conductance (MscS) and the ATP-dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function.

FEBS Lett, 2003 Nov 27, 555(1), 96 - 101
The lactose permease of Escherichia coli: overall structure, the sugar-binding site and the alternating access model for transport; Abramson J et al.; Membrane transport proteins transduce free energy stored in electrochemical ion gradients into a concentration gradient and are a major class of membrane proteins, many of which play important roles in human health and disease . Recently, the X-ray structure of the Escherichia coli lactose permease (LacY), an intensively studied member of a large group of related membrane transport proteins, was solved at 3.5 A . LacY is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the molecule . The structure represents the inward-facing conformation, as evidenced by a large internal hydrophilic cavity open to the cytoplasmic side . The structure with a bound lactose homolog reveals the sugar-binding site in the cavity, and a mechanism for translocation across the membrane is proposed in which the sugar-binding site has alternating accessibility to either side of the membrane.

FEBS Lett, 2003 Nov 27, 555(1), 79 - 84
Selectivity and conductance among the glycerol and water conducting aquaporin family of channels; Stroud RM et al.; The atomic structures of a transmembrane water plus glycerol conducting channel (GlpF), and now of aquaporin Z (AqpZ) from the same species, Escherichia coli, bring the total to three atomic resolution structures in the aquaporin (AQP) family . Members of the AQP family each assemble as tetramers of four channels . Common helical axes support a wider channel in the glycerol plus water channel paradigm, GlpF . Water molecules form a single hydrogen bonded file throughout the 28 A long channel in AqpZ . The basis for absolute exclusion of proton or hydronium ion conductance through the line of water is explored using simulations.

FEBS Lett, 2003 Nov 27, 555(1), 29 - 34
Mechanics of coupling proton movements to c-ring rotation in ATP synthase; Fillingame RH et al.; F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane . Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane . Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61 . Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs) . Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface . We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle . The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.

Reprod Domest Anim, 2003 Dec, 38(6), 464 - 9
Peripheral blood neutrophil function and lymphocyte subpopulations in cycling mares; Roberto Da Costa RP et al.; The purpose of this study was to evaluate different parameters of the immune status in the mare, during the follicular and the luteal phases of the oestrous cycle, in two consecutive years . Functional competence of peripheral blood neutrophils, such as chemotaxis, phagocytosis and oxidative burst was assessed under physiological cyclic conditions (Exp . I) . In the second year of this study (Exp . II), besides peripheral blood neutrophil phagocytosis and oxidative burst analysis, circulating lymphocyte subsets were also characterized . The reproductive status in a total of 17 adult cycling mares was evaluated by ultrasonography and further confirmed by plasma progesterone levels . Chemotaxis tests were performed using porous membranes inserted in transwell chambers . Lipopolysaccharide (LPS) from Escherichia coli and N-formyl-Met-Leu-Phe (fMLP) were used as chemoattractants . Measurement of phagocytosis and oxidative burst in blood neutrophils were assessed by flow cytometry using commercially available kits . Quantification of T-lymphocyte subsets was assessed by indirect immunofluorescence staining after incubation with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers by flow cytometry . Natural killer cells and B cells were estimated mathematically . No significant difference was found in migration, phagocytosis and oxidative burst at either phase of the oestrous cycle . Statistical analysis of total white blood cell counts also showed no significant difference between either phase of the oestrous cycle, although there was a tendency for blood neutrophils to increase in number under the progesterone influence (p = 0.09) . Lymphocytic subpopulations did not differ throughout the oestrous cycle . Overall, our results suggest that luteal and follicular phases in cycling mares may not influence the immune status of the mare.

Biochem J, 2004 Mar 1, 378(Pt 2), 529 - 38
Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax; Na BK et al.; Cysteine proteases play important roles in the life cycles of malaria parasites . Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets . To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P . vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli . The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain . Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2 . Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains . Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(4), 230 - 3
{Cloning, expression and purification of the major surface antigen P30 of Toxoplasma gondii}; Zheng DL et al.; OBJECTIVE: To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecular cloning . METHODS: The gene of P30 (containing the whole P30 gene sequence, without the gene encoding signal peptide) was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence of P30 . The recombinant plasmid was constructed using EcoR I, Xho I and was then transformed into E . coli Top10 . The positive clones were identified by restriction enzymes and DNA sequence analysis . The fusion protein was induced by IPTG and purified by affinity chromatography using ProBond Resin (a kind of nickel-charged sepharose resin) and was identified by SDS-PAGE and Western blotting . RESULTS: The products of PCR, cleavage and link reaction were same as expected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy mutation . A 58 kDa fusion protein was induced by IPTG and was purified by chromatography . CONCLUSION: Fusion protein containing Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(3), 144 - 6
{Cloning and identification of the gene fragments of Paragonimus westermani}; Ling JJ et al.; OBJECTIVE: To screen and identify the recombinants from the cDNA library of the adult Paragonimus westermani(PwA) for immunodiagnosis and immunoprophylaxis . METHODS: PwA cDNA library was screened with the PwA antigen immunized rabbit sera(IRS) pre-absorbed by the extract of E . coli XL1-Blue . The recombinants from positive clones were amplified by PCR, sequenced and cut off by KpmI/BamHI and, then sub-cloned into pRESETB vector . The fusion protein was expressed, analysed by SDS-PAGE and identified by Western blotting with immune rabbit serum against worm antigen of Paragonimus westermani . RESULTS: The inserted cDNA fragment from the positive clone Pw-2 was about 800 bp, which contained an open reading frame(ORF) encoding Pw pre-procathepsin L belonging to cysteinase family . Expression product of Pw-2 was a fusion protein of 32 kDa, which can be recognized by immune rabbit serum against worm antigen of Paragominus westermani . CONCLUSION: A recombinant plasmid Pw-2 encodes Pw pre-procathepsin L is constructed.

Nucleic Acids Res . 2003 Dec 1;31(23):e152.
Suppression of gene expression by a cell-permeable Tet repressor; Mortlock A et al.; Engineered transcription factors designed to selectively activate or repress endogenous genes have great potential in medical and biotechnological applications . Ultimately, their success will depend on the development of efficient delivery systems . We show here that a chimeric tetracycline- controlled transcription factor, encompassing the Tet repressor (TetR) from the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and a cell membrane transducing peptide, is able to regulate transcription from a tetracycline responsive promoter (pCMV2xtetO2) . When added directly to cultured cells, TetR fused to the full-length Antennapedia homeodomain (AntpHD) from Drosophila (TetRAntp), was able to selectively repress transcription in cells transiently transfected with a tetracycline-regulated reporter transcription unit . Moreover, TetRAntp could repress expression of a tetracycline responsive reporter transcription unit stably integrated into the genome of HeLa cells, demonstrating the possibility of manipulating endogenous gene expression by cell-permeable transcription factors.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6976 - 85
Unexpected correlations between gene expression and codon usage bias from microarray data for the whole Escherichia coli K-12 genome; dos Reis M et al.; Escherichia coli has long been regarded as a model organism in the study of codon usage bias (CUB) . However, most studies in this organism regarding this topic have been computational or, when experimental, restricted to small datasets; particularly poor attention has been given to genes with low CUB . In this work, correspondence analysis on codon usage is used to classify E.coli genes into three groups, and the relationship between them and expression levels from microarray experiments is studied . These groups are: group 1, highly biased genes; group 2, moderately biased genes; and group 3, AT-rich genes with low CUB . It is shown that, surprisingly, there is a negative correlation between codon bias and expression levels for group 3 genes, i.e . genes with extremely low codon adaptation index (CAI) values are highly expressed, while group 2 show the lowest average expression levels and group 1 show the usual expected positive correlation between CAI and expression . This trend is maintained over all functional gene groups, seeming to contradict the E.coli-yeast paradigm on CUB . It is argued that these findings are still compatible with the mutation-selection balance hypothesis of codon usage and that E.coli genes form a dynamic system shaped by these factors.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6852 - 9
Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors; Klumpp K et al.; Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT) . RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved . We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions . Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+ . Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding . Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves . Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations . These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases . In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism . Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6828 - 40
Functional characterization of Helicobacter pylori DnaB helicase; Soni RK et al.; Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans . Not much is known regarding DNA replication in H.pylori that is important for cell survival . Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo . Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively . HpDnaB showed strong 5' to 3' helicase and ATPase activity . Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC) . However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase . Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other . It is possible that a functional DnaC homolog is present in H.pylori . The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6806 - 18
The common and the distinctive features of the bulged-G motif based on a 1.04 A resolution RNA structure; Correll CC et al.; Bulged-G motifs are ubiquitous internal RNA loops that provide specific recognition sites for proteins and RNAs . To establish the common and distinctive features of the motif we determined the structures of three variants and compared them with related structures . The variants are 27-nt mimics of the sarcin/ricin loop (SRL) from Escherichia coli 23S ribosomal RNA that is an essential part of the binding site for elongation factors (EFs) . The wild-type SRL has now been determined at 1.04 A resolution, supplementing data obtained before at 1.11 A and allowing the first calculation of coordinate error for an RNA motif . The other two structures, having a viable (C2658U*G2663A) or a lethal mutation (C2658G*G2663C), were determined at 1.75 and 2.25 A resolution, respectively . Comparisons reveal that bulged-G motifs have a common hydration and geometry, with flexible junctions at flanking structural elements . Six conserved nucleotides preserve the fold of the motif; the remaining seven to nine vary in sequence and alter contacts in both grooves . Differences between accessible functional groups of the lethal mutation and those of the viable mutation and wild-type SRL may account for the impaired elongation factor binding to ribosomes with the C2658G*G2663C mutation and may underlie the lethal phenotype.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6700 - 9
An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli; Santoro SW et al.; The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli . The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon . Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair . The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences . The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii . The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli . Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.

Protein Sci, 2003 Dec, 12(12), 2838 - 43
Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC; Lesniak J et al.; The osmotically inducible protein OsmC, like its better-characterized homolog, the organic hydroperoxide protein Ohr, is involved in defense against oxidative stress caused by exposure to organic hydroperoxides . The crystal structure of Escherichia coli OsmC reported here reveals that the protein is a tightly folded domain-swapped dimer with two active sites located at the monomer interface on opposite sides of the molecule . We demonstrate that OsmC preferentially metabolizes organic hydroperoxides over inorganic hydrogen peroxide . On the basis of structural and enzymatic similarities, we propose that the OsmC catalytic mechanism is analogous to that of the Ohr proteins and of the structurally unrelated peroxiredoxins, directly using highly reactive cysteine thiol groups to elicit hydroperoxide reduction.

Protein Sci, 2003 Dec, 12(12), 2748 - 56
Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily; Lemieux MJ et al.; Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane . GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily . It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane . Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT . Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity . Nine such detergents were later used for GlpT crystallization . Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range . Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals . These crystals exhibited order to 3.7 A resolution in two dimensions . However, the stacking in the third dimension was partially disordered . This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution . The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.

J Biol Chem, 2004 Jan 9, 279(2), 1330 - 5 Epub 2003 Nov 19.
Reconstitution of coat protein complex II (COPII) vesicle formation from cargo-reconstituted proteoliposomes reveals the potential role of GTP hydrolysis by Sar1p in protein sorting; Sato K et al.; Secretory proteins are transported from the endoplasmic reticulum (ER) in vesicles coated with coat protein complex II (COPII) . To investigate the molecular mechanism of protein sorting into COPII vesicles, we have developed an in vitro budding reaction comprising purified coat proteins and cargo reconstituted proteolipsomes . Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER . Recombinant Emp46/47p proteins and the ER resident protein Ufe1p were reconstituted into liposomes whose composition resembles yeast ER membranes . When the proteoliposomes were mixed with COPII proteins and GMP-PNP, Emp46/47p, but not Ufe1p, were concentrated into COPII vesicles . We also show here that reconstituted Emp47p accelerates the GTP hydrolysis by Sar1p as stimulated by its GTPase-activating protein, Sec23/24p, both of which are components of the COPII coat . Furthermore, this GTP hydrolysis decreases the error of cargo sorting . We suggest that GTP hydrolysis by Sar1p promotes exclusion of improper proteins from COPII vesicles.

J Biol Chem, 2004 Feb 6, 279(6), 4604 - 11 Epub 2003 Nov 19.
Different contributions of the three CXXC motifs of human protein-disulfide isomerase-related protein to isomerase activity and oxidative refolding; Horibe T et al.; Human protein-disulfide isomerase (hPDI)-related protein (hPDIR), which we previously cloned from a human placental cDNA library (Hayano, T., and Kikuchi, M . (1995) FEBS Lett . 372, 210-214), and its mutants were expressed in the Escherichia coli pET system and purified by sequential nickel affinity resin chromatography . Three thioredoxin motifs (CXXC) of purified hPDIR were found to contribute to its isomerase activity with a rank order of CGHC > CPHC > CSMC, although both the isomerase and chaperone activities of this protein were lower than those of hPDI . Screening for hPDIR-binding proteins using a T7 phage display system revealed that alpha1-antitrypsin binds to hPDIR . Surface plasmon resonance experiments demonstrated that alpha1-antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5) . Interestingly, the rate of oxidative refolding of alpha1-antitrypsin with hPDIR was much higher than with hPDI or hP5 . Thus, the substrate specificity of hPDIR differed from that associated with isomerase activity, and the contribution of the CSMC motif to the oxidative refolding of alpha1-antitrypsin was the most definite of the three (CSMC, CGHC, CPHC) . Substitution of SM and PH in the CXXC motifs with GH increased isomerase activity and decreased oxidative refolding . In contrast, substitution of GH and PH with SM decreased isomerase activity and increased oxidative refolding . Because CXXC motif mutants lacking isomerase activity retain chaperone activity for the substrate rhodanese, it is clear that, similar to PDI and hP5, the isomerase and chaperone activities of hPDIR are independent . These results suggest that the central dipeptide of the CXXC motif is critical for both redox activity and substrate specificity.

Acta Pharmacol Sin, 2003 Nov, 24(11), 1108 - 12
Cloning and gene expression of G protein competitive inhibitory polypeptide and its prophylactic effects on myocardial hypertrophy in vitro; Zhou JZ et al.; AIM: To clone and express G protein competitive inhibitory polypeptide (GCIP) gene and investigate the prophylactic effects of GCIP on myocardial hypertrophy in vitro . METHODS: The pIVEX2.3MCS-GCIP plasmid expressing GCIP was constructed by inserting double-stranded oligonucleotide into pIVEX2.3-MCS plasmid vector . Recombinant plasmid was expressed in Rapid Translation System 500 (RTS500) . The expression of GCIP was identified by SDS-PAGE and Western blotting . The purification of GCIP with 6xHis tag was carried out on a Ni-NTA agarose column . The prophylactic effects of GCIP was observed on cardiomyocytes isolated from newborn Wistar rats . Protein synthesis rate were assessed by {3H}Leu incorporation . Protein contents were measured by Lowry method . RESULTS: The plasmid pIVEX2.3MCS-GCIP was successfully constructed . The expression of GCIP was about 2.43 % of total protein . GCIP was purified on Ni-NTA agarose column . The purity of the purified GCIP peptide was about 98 % . The contents of total protein and the rate of {3H}Leu incorporation were significantly decreased in (100 microg/L, 1 mg/L, and 10 mg/L) GCIP-treated groups in myocardial cellular hypertrophy model (P<0.01) . CONCLUSION: The pIVEX2.3MCS-GCIP expression vector has been constructed successfully . The GCIP was expressed in RTS500 system and was purified with Ni-NTA agarose . GCIP was able to inhibit myocardial hypertrophy concentration-dependently in vitro.

Biotechnol Lett, 2003 Oct, 25(20), 1751 - 5
Production of 5-aminolevulinic acid by an Escherichia coli aminolevulinate dehydratase mutant that overproduces Rhodobacter sphaeroides aminolevulinate synthase; Xie L et al.; Two Escherichia coli strains, a wild-type strain and a hemB mutant, which contained the hemA gene from Rhodobacter sphaeroides produced aminolevulinate at 4 g l(-1) . The hemB mutant did not increase the accumulation of aminolevulinate, an observation attributed to its 50% lower aminolevulinate synthase activity in than the wild-type . Growth was limited for both strains probably due to the accumulation of 0.5 g aminoacetone l(-1).

Biotechnol Lett, 2003 Oct, 25(20), 1729 - 34
Refolding of a recombinant full-length non-structural (NS3) protein from hepatitis C virus by chromatographic procedures; Li M et al.; The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) was expressed as inactive aggregates in Escherichia coli . The protein was refolded by chromatographic techniques of which ion exchange chromatography was best for crude samples and gel filtration best for partially purified samples . Immobilized metal ion affinity chromatography showed intermediate performance . Gradient procedures enhanced the recovery of active NS3 protein.

Biotechnol Lett, 2003 Oct, 25(20), 1695 - 701
Surface charge engineering of PQQ glucose dehydrogenase for downstream processing; Koh H et al.; The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification . The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH) . During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme . After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample . Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability . Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process.

Proteomics, 2003 Oct, 3(10), 1912 - 9
Overcoming technical variation and biological variation in quantitative proteomics; Molloy MP et al.; Quantitative proteomics investigates physiology at the molecular level by measuring relative differences in protein expression between samples under different experimental conditions . A major obstacle to reliably determining quantitative changes in protein expression is to overcome error imposed by technical variation and biological variation . In drug discovery and development the issue of biological variation often rises in concordance with the developmental stage of research, spanning from in vitro assays to clinical trials . In this paper we present case studies to raise awareness to the issues of technical variation and biological variation and the impact this places on applying quantitative proteomics . We defined the degree of technical variation from the process of two-dimensional electrophoresis as 20-30% coefficient of variation . On the other hand, biological variation observed experiment-to-experiment showed a broader degree of variation depending upon the sample type . This was demonstrated with case studies where variation was monitored across experiments with bacteria, established cell lines, primary cultures, and with drug treated human subjects . We discuss technical variation and biological variation as key factors to consider during experimental design, and offer insight into preparing experiments that overcome this challenge to provide statistically significant outcomes for conducting quantitative proteomic research.

Shock, 2003 Dec, 20(6), 551 - 7
Reconstituted high-density lipoprotein attenuates organ injury and adhesion molecule expression in a rodent model of endotoxic shock; McDonald MC et al.; The salutary effects of high-density lipoproteins (HDLs) in animal and human models of endotoxic shock have in the past been attributed to the ability of this lipoprotein to bind to lipopolysaccharide . However, the precise mechanisms for the protective effect of HDL are unclear . The first objective of this study was to determine the effects of HDLs on the organ injury and dysfunction associated with acute severe endotoxemia . Second, to gain insight into the mechanism of action of HDL, we also investigated the effect of HDLs on 1) the expression of P-selectin and intercellular adhesion molecule-1 in the kidneys of rats treated with endotoxin and 2) the rise in the plasma levels of tumor necrosis factor-alpha (TNF-alpha) . Rats were given Escherichia coli lipopolysaccharide (6 mg/kg i.v.), pretreated with either vehicle (n = 9) or reconstituted HDL (rHDL; apolipoprotein A-I/phosphatidylcholine proteoliposomes, n = 10), and were monitored for 6 h . Here we report that rHDL attenuates the renal injury and dysfunction caused by endotoxin in the rat . In addition, rHDL reduced the degree of histological tissue injury in the lung, liver and intestine and attenuated the expression of P-selectin and intercellular adhesion molecule-1 in the renal glomerulus . Interestingly, pretreatment of rats with rHDL did not prevent the hypotension nor the rise in plasma levels of TNF-alpha (at 90 min) caused by endotoxin . Thus, rHDL reduces the organ injury/dysfunction, but does not affect the circulatory failure, nor the rise in plasma levels of TNF-alpha caused by endotoxin in the rat . We propose that the mechanisms of these beneficial effects of HDL may be related to direct inhibition of adhesion molecule expression.

J Biol Chem, 2004 Feb 13, 279(7), 5216 - 26 Epub 2003 Nov 18.
Hypo-osmotic stress activates Plc1p-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol Hexakisphosphate accumulation in yeast; Perera NM et al.; Polyphosphoinositide-specific phospholipases (PICs) of the delta-subfamily are ubiquitous in eukaryotes, but an inability to control these enzymes physiologically has been a major obstacle to understanding their cellular function(s) . Plc1p is similar to metazoan delta-PICs and is the only PIC in Saccharomyces cerevisiae . Genetic studies have implicated Plc1p in several cell functions, both nuclear and cytoplasmic . Here we show that a brief hypo-osmotic episode provokes rapid Plc1p-catalyzed hydrolysis of PtdIns(4,5)P2 in intact yeast by a mechanism independent of extracellular Ca2+ . Much of this PtdIns(4,5)P2 hydrolysis occurs at the plasma membrane . The hydrolyzed PtdIns(4,5)P2 is mainly derived from PtdIns4P made by the PtdIns 4-kinase Stt4p . PtdIns(4,5)P2 hydrolysis occurs normally in mutants lacking Arg82p or Ipk1p, but they accumulate no InsP6, showing that these enzymes normally convert the liberated Ins(1,4,5)P3 rapidly and quantitatively to InsP6 . We conclude that hypo-osmotic stress activates Plc1p-catalyzed PtdIns(4,5)P2 at the yeast plasma membrane and the liberated Ins(1,4,5)P3 is speedily converted to InsP6 . This ability routinely to activate Plc1p-catalyzed PtdIns(4,5)P2 hydrolysis in vivo opens up new opportunities for molecular and genetic scrutiny of the regulation and functions of phosphoinositidases C of the delta-subfamily.

J Biol Chem, 2004 Feb 20, 279(8), 6434 - 43 Epub 2003 Nov 18.
Sticky DNA formation in vivo alters the plasmid dimer/monomer ratio; Vetcher AA et al.; Our discovery that plasmids containing the Friedreich's ataxia (FRDA) expanded GAA.TTC sequence, which forms sticky DNA, are prone to form dimers compared with monomers in vivo is the basis of an intracellular assay in Escherichia coli for this unusual DNA conformation . Sticky DNA is a single long GAA.GAA.TTC triplex formed in plasmids harboring a pair of long GAA.TTC repeat tracts in the direct repeat orientation . This requirement is fulfilled by either plasmid dimers of DNAs with a single trinucleotide repeat sequence tract or by monomeric DNAs containing a pair of direct repeat GAA.TTC sequences . DNAs harboring a single GAA.TTC repeat are unable to form this type of triplex conformation . An excellent correlation was observed between the ability of a plasmid to adopt the sticky triplex conformation as assayed in vitro and its propensity to form plasmid dimers relative to monomers in vivo . The variables measured that strongly influenced these measurements are as follows: length of the GAA.TTC insert; the extent of periodic interruptions within the repeat sequence; the orientation of the repeat inserts; and the in vivo negative supercoil density . Nitrogen mustard cross-linking studies on a family of GAA.TTC-containing plasmids showed the presence of sticky DNA in vivo and, thus, serves as an important bridge between the in vitro and in vivo determinations . Biochemical genetic studies on FRDA containing DNAs grown in recA or nucleotide excision repair or ruv-deficient cells showed that the in vivo properties of sticky DNA play an important role in the monomer-dimer-sticky DNA intracellular intercon-versions . Thus, the sticky DNA triplex exists and functions in living cells, strengthening the likelihood of its role in the etiology of FRDA.

J Biol Chem, 2004 Jan 30, 279(5), 3492 - 6 Epub 2003 Nov 18.
RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli; Chow KH et al.; In Escherichia coli, recF and recR are required to stabilize and maintain replication forks arrested by UV-induced DNA damage . In the absence of RecF, replication fails to recover, and the nascent lagging strand of the arrested replication fork is extensively degraded by the RecQ helicase and RecJ nuclease . recO mutants are epistatic with recF and recR with respect to recombination and survival assays after DNA damage . In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks after UV-irradiation . In the absence of RecO, the nascent DNA at arrested replication forks is extensively degraded and replication fails to recover . The extent of nascent DNA degradation is equivalent in single, double, or triple mutants of recF, recO, or recR, and the degradation is dependent upon RecJ and RecQ functions . Because RecF has been shown to protect the nascent lagging strand from degradation, these observations indicate that RecR and RecO function with RecF to protect the same nascent strand of the arrested replication fork and are likely to act at a common point during the recovery process . We discuss these results in relation to the biochemical and cellular properties of RecF, RecO, and RecR and their potential role in loading RecA filaments to maintain the replication fork structure after the arrest of replication by UV-induced DNA damage.

J Biol Chem, 2004 Jan 30, 279(5), 3484 - 91 Epub 2003 Nov 18.
Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2; Pandey KC et al.; The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target . Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme . Rather, folding is mediated by an amino-terminal extension of the mature protease . As in related enzymes, the prodomain is a potent inhibitor of falcipain-2 . We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases . The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr . Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily . Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide . Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound . The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin . The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved . Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding . Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition.

J Biol Chem, 2004 Feb 20, 279(8), 6444 - 54 Epub 2003 Nov 18.
Structure-dependent recombination hot spot activity of GAA.TTC sequences from intron 1 of the Friedreich's ataxia gene; Napierala M et al.; The recombinational properties of long GAA.TTC repeating sequences were analyzed in Escherichia coli to gain further insights into the molecular mechanisms of the genetic instability of this tract as possibly related to the etiology of Friedreich's ataxia . Intramolecular and intermolecular recombination studies showed that the frequency of recombination between the GAA.TTC tracts was as much as 15 times higher than the non-repeating control sequences . Homologous, intramolecular recombination between GAA.TTC tracts and GAAGGA.TCCTTC repeats also occurred with a very high frequency (approximately 0.8%) . Biochemical analyses of the recombination products demonstrated the expansions and deletions of the GAA.TTC repeats . These results, together with our previous studies on the CTG.CAG sequences, suggest that the recombinational hot spot characteristics may be a common feature of all triplet repeat sequences . Unexpectedly, we found that the recombination properties of the GAA.TTC tracts were unique, compared with CTG.CAG repeats, because they depended on the DNA secondary structure polymorphism . Increasing the length of the GAA.TTC repeats decreased the intramolecular recombination frequency between these tracts . Also, a correlation was found between the propensity of the GAA.TTC tracts to adopt the sticky DNA conformation and the inhibition of intramolecular recombination . The use of novobiocin to modulate the intracellular DNA topology, i.e . the lowering of the negative superhelical density, repressed the formation of the sticky DNA structure, thereby restoring the expected positive correlation between the length of the GAA.TTC tracts and the frequency of intramolecular recombination . Hence, our results demonstrate that sticky DNA exists and functions in E . coli.

Chem Phys Lipids, 2003 Sep, 125(1), 27 - 39
Nuclear magnetic resonance of lipid A--the influence of solvents on spin relaxation and spectral quality; Brecker L; Nuclear magnetic resonance (NMR) spectroscopy of lipid A is limited by rapid transversal relaxation and subsequent line broadening caused by the tendency of these glycolipids to form aggregates in all solvents . To examine the influence of solvents on NMR spectra, hexa-acyl lipid A from Escherichia coli F515 was investigated . Line widths at half height, longitudinal relaxation times, and transversal relaxation times were measured in different solvents, lipid A concentrations, and temperatures . Chloroform-d, dioxane-d(8), and pyridine-d(5) each mixed with 25% methanol-d(4) as well as sole DMSO-d(6) and 0.1M triethylamine-d(15) (TEA-d(15)) in D(2)O caused good spectral resolutions and allowed structure analysis . ROESY and HMBC spectra gave an insight into the influence of transversal relaxation times on spectral quality in two-dimensional spectra . Solvent depending differences of interglycosidic NOEs indicated dissimilarities of the conformations in the interglycosidic linkage and allowed conclusions about the lipid A solution state.

J Insect Physiol, 2003 Dec, 49(12), 1199 - 209
Probiotic effects of beta-glucuronidase on the peach-potato aphid Myzus persicae (Aphididae); Cherqui A et al.; beta-glucuronidase (GUS) is a reporter protein commonly expressed in transgenic plants allowing the visualization of the transformed individuals . In our recent work, we showed that consumption of transformed potato plants expressing this GUS enzyme improves performance of the phloem feeding aphid Myzus persicae . Those results led us to the conclusion that the expression of GUS in potato plants might be responsible for the probiotic effect measured in feeding aphids . In the present paper, artificial diets were used to provide active GUS (10 and 500 microg ml(-1)), inactivated heated GUS (500 microg ml(-1)), glucuronic acid (10, 100 and 500 microg ml(-1)), and bovine serum albumin (500 microg ml(-1)) to M . persicae . Our results reveal that these chemicals provided as food intake might influence the biological parameters of this aphid . Experiments showed a probiotic effect of 500 microg ml(-1) GUS diet, resulting in reduced larval mortality, and increased adult reproduction period and fecundity, which led to an increased population growth potential (r(m)=0.17+/-0.01 versus r(m)=0.12+/-0.03 for aphids fed on control diet) . A lower amount of added GUS led to fewer variations, biological parameters being only slightly altered (r(m)=0.14+/-0.03) . Statistically similar alterations of the biological parameters were obtained when comparing aphids fed on the diet added with inactivated GUS or the non-structural bovine serum albumin protein (r(m)=0.15+/-0.02 and 0.14+/-0.03, respectively) . Feeding assays conducted with glucuronic acid supplemented diets enhanced longevity and nymph production of the adult aphids and reduced larval mortality, resulting in r(m)=0.15+/-0.02 for the highest dose (500 microg ml(-1)) . Although 100 microg ml(-1) glucuronate diet did not induce any effect on M . persicae (r(m)=0.12+/-0.03), aphids fed on 10 microg ml(-1) glucuronate diet exhibited unexpected reduced demographic parameters (r(m)=0.10+/-0.03) . Immuno-histological analysis showed GUS labeling along the whole digestive epithelium of adults and in various tissues including embryos and bacteriocytes . These results suggest that GUS crosses through the digestive tract . Western blots performed with protein extracts of transformed potato plants expressing the gus gene showed a unique band of molecular weight 76 kDa . On the contrary, in extracts from aphids fed on transgenic potato plants or bred on GUS 500 microg ml(-1) artificial diet, several proteins of lower molecular weight were hybridized, revealing proteolysis of ingested GUS . It is concluded that GUS protein, and more precisely GUS activity, is responsible for the probiotic effects on aphid feeding . The possible pathways of induction of such physiological alterations by GUS are discussed.

J Insect Physiol, 2003 Dec, 49(12), 1135 - 44
Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust; Hayward DC et al.; The ecdysone receptor(1), which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths . It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts . Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here . Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation . After expression in E . coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A . Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains . In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts . Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III . Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

Methods, 2004 Jan, 32(1), 21 - 4
Hsp-antigen fusion and their use for immunization; Udono H et al.; Immunization with antigenic peptide non-covalently associated with HSP elicits the peptide specific CD8+T cell response . The evidence encourages us to test the vaccination effect of recombinant HSP to which antigenic peptides are genetically fused . In the fusion protein, there should be no empty HSP molecules that failed to associate with the peptide of interest, like in vitro reconstitution method, therefore, promising effect may be easily obtained . Recombinant proteins expressed in Escherichia coli often form inclusion bodies and are thereby obtained as insoluble proteins or as proteins lacking their original functions . We describe here a simple and rapid refolding method of histidine-tagged recombinant hsp70/hsp70-peptide complex using a Ni(2+)-agarose column chromatography, without taking a process of dialysis to remove denaturants . The hsp70(hsp70-peptide complex) expressed in E . coli as a form of inclusion body was solubilized in 8 M urea containing buffer and applied to a Ni(2+)-agarose column . The bound hsp70 was refolded on the column by quick removal of urea with urea-free buffer and eluted with a denaturant-free and imidazole-containing buffer . The purified hsp70 was homogeneous and soluble . In addition, it had a very high ATPase activity and strong CTL inducing activity, whereas hsp70 prepared by conventional dialysis method had a negligible ATPase activity . This simple and rapid refolding method may provide a general method for a restoration of function (and/or immunization effect) and solubility of histidine-tagged recombinant HSP.

Bioconjug Chem, 2003 Nov-Dec, 14(6), 1243 - 52
Oligomeric assembly and ligand binding of the members of protein family YER057c/YIL051c/YJGF; Mistiniene E et al.; Proteins UK114 and p14.5 are both members of the putative family of small proteins YER057c/YIL051c/YjgF . The biological role of these proteins is not understood very well, and in addition, their oligomeric structure in solution remains controversial . We therefore investigated the oligomeric structure of UK114 and p14.5 using a number of methods . Both proteins have exhibited a homotrimeric structure in solution . Indeed the trimeric structure of the two proteins appeared to be so similar that when protein subunits derived from different species were mixed, stable heterotrimeric complexes (monomer ratio of 1:2 and 2:1 of UK114 and p14.5, respectively) could be formed in vitro . Furthermore, the trimeric structure of both UK114 and p14.5 proved essential for the stoichiometric hydrophobic ligand, such as fatty acid binding activity of the two proteins.

J Am Chem Soc, 2003 Nov 26, 125(47), 14307 - 12
Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA:ACP transacylase; Kumar P et al.; Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains . The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module . New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues . We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides . Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B . The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.

RNA, 2003 Dec, 9(12), 1437 - 45
Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA; Childs JL et al.; Oligonucleotide directed misfolding of RNA (ODMiR) uses short oligonucleotides to inhibit RNA function by exploiting the ability of RNA to fold into different structures with similar free energies . It is shown that the 2'-O-methyl oligonucleotide, m(CAGCCUACCCGG), can trap Escherichia coli RNase P RNA (M1 RNA) in a nonfunctional structure in a transcription mixture containing RNase P protein (C5 protein) . At about 200 nM, the 12-mer thus inhibits 50% of pre-tRNA processing by RNase P . Roughly 10-fold more 12-mer is required to inhibit RNase P containing full-length, renatured RNase P RNA . Diethyl pyrocarbonate modification in the presence of 12-mer reveals increased modification of sites in and interacting with P4, suggesting a structural rearrangement of a large pseudoknot important for catalytic activity . Thus, the ODMiR method can be applied to RNAs even when folding is facilitated by a cognate protein.

RNA, 2003 Dec, 9(12), 1418 - 21
Demonstration of the role of the DnaK chaperone system in assembly of 30S ribosomal subunits using a purified in vitro system; Maki JA et al.; Recently, there has been controversy regarding the ability of the DnaK chaperone system to facilitate Escherichia coli 30S subunit assembly at otherwise nonpermissive conditions . Here, we present additional data indicating that purified DnaK chaperone assembled 30S subunits are functional . Additionally, explanations for reported differences are discussed.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14719 - 24 Epub 2003 Nov 17.
The thermodynamics of template-directed DNA synthesis: base insertion and extension enthalpies; Minetti CA et al.; We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension . Specifically, we used families of hairpin self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues . Our results reveal exothermic heats between -9.8 and -16.0 kcal/bp for template-directed enzymatic polymerization . These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base . Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies . We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes . It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process . Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template-primer level . Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.

J Histochem Cytochem, 2003 Dec, 51(12), 1655 - 64
Localization of a brain sulfotransferase, SULT4A1, in the human and rat brain: an immunohistochemical study; Liyou NE et al.; Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs . To date, four cytosolic sulfotransferases have been shown to be expressed in human brain . Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown . Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1 . These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain . In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem . Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen . The findings from this study may assist in determining the physiological role of this SULT isoform.

J Biol Chem, 2004 Jan 30, 279(5), 3434 - 8 Epub 2003 Nov 17.
Mouse liver CYP2C39 is a novel retinoic acid 4-hydroxylase . Its down-regulation offers a molecular basis for liver retinoid accumulation and fibrosis in aryl hydrocarbon receptor-null mice; Andreola F et al.; Livers of aryl hydrocarbon receptor (AHR)-null mice have high levels of retinoic acid (RA), retinol, and retinyl palmitate . Hepatic accumulation of RA in these mice may be responsible in part for the hepatic phenotype characterized by small liver size and fibrosis . The increased levels of hepatic RA may be due to decreased metabolism of RA to 4-hydroxyretinoic acid . To identify the P450 isoform(s) involved in RA metabolism, liver microsomes from AHR-null and wild-type mice were subjected to Western blotting and probed with antibodies to rat P450s that cross-react with murine forms . Signal intensity in Western blots probed with anti-rat CYP2C6 antibodies correlated with levels of RA 4-hydroxylation . Furthermore, this anti-rat CYP2C6 antibody inhibited RA 4-hydroxylase activity of wild-type mouse liver microsomes to the levels of AHR-null mouse liver . When used to screen a mouse liver cDNA expression library, this antibody exclusively recognized the murine P450 CYP2C39 . Catalytic assays of five recombinant mouse CYP2Cs expressed in Escherichia coli revealed that only CYP2C39 was competent for RA 4-hydroxylation (K(m) = 812.3 nm and V(max) 47.85 (fmol/min/pmol P450)) . Real time reverse transcriptase-PCR used to assess the Cyp2C39 mRNA expression showed decreased levels (30%) of this transcript in AHR-null compared with wild-type liver, consistent with decreased protein levels observed by Western blot analysis using an antibody to a CYP2C39-specific peptide . These data show that CYP2C39 catalyzes RA catabolism and thus possibly controls RA levels in mouse liver . Down-regulation of Cyp2C39 is hypothesized to be responsible for the liver phenotype in the AHR-null mouse.

J Steroid Biochem Mol Biol, 2003 Sep, 86(3-5), 231 - 7
Structure-function studies of aromatase and its inhibitors: a progress report; Chen S et al.; The utilization of computer modeling, site-directed mutagenesis, inhibition kinetic analysis and reaction metabolite analysis allows us to better understand the structure-function relationship between aromatase and its inhibitors . Our results have helped in determining how steroidal and nonsteriodal aromatase inhibitors bind to the active site of the enzyme . This information has also aided in the understanding of the reaction mechanism of aromatase . Furthermore, our structure-function studies of aromatase have generated important information for predicting how environmental chemicals interact with the enzyme . During the last 2 years, a new aromatase computer model based on the X-ray structure of rabbit cytochrome P450 2C5 has been generated and used to evaluate the results obtained from new aromatase mutants produced in this laboratory . In addition, we have succeeded in the expression and purification of functionally active aromatase using an Escherichia coli expression method . The catalytic properties of this recombinant aromatase are similar to those properties exhibited by the human placental aromatase preparation and the mammalian cell-expressed enzyme . The E . coli expressed aromatase will be very useful for further structure-function studies of aromatase . Our laboratory has also evaluated the growth-inhibiting activity of aromatase inhibitors in estrogen receptor-positive breast cancer using three-dimensional cell cultures of aromatase-over expressing MCF-7 and T-47D cell lines (i.e . MCF-7aro and T-47Daro) . Our results demonstrate that these three-dimensional cultures are valuable approaches to assess the growth-inhibiting activity of aromatase inhibitors . Finally, we have identified several phytochemicals to be potent inhibitors of aromatase . To demonstrate the impact of the phytochemicals on estrogen formation in vivo, we showed that the intake of anti-aromatase chemicals from red wine was capable of suppressing MCF-7aro-mediated tumor formation in nude mice and aromatase-induced hyperplasia in a transgenic mouse model in which aromatase is over-expressed in the mammary tissue.

Biochem Biophys Res Commun, 2003 Nov 21, 311(3), 685 - 90
Human connective tissue growth factor expressed in Escherichia coli is a non-mitogenic inhibitor of apoptosis; Gygi D et al.; After cloning and sequencing chicken connective tissue growth factor (cCTGF) we showed that in serum-deprived chicken embryo fibroblasts (CEF) the initial decline of cCTGF mRNA was followed by an increase . Human CTGF that was produced as a soluble protein in Escherichia coli by thioredoxin co-expression inhibited serum-deprivation induced apoptosis of CEF at I(50) of approximately 8pM, demonstrating a function of CTGF at concentrations observed in supernatants of fibroblasts . CTGF was neither mitogenic for CEF nor for other cell types . In conclusion CTGF supports very efficiently survival of certain cells without stimulating their cell growth.

Biochem Biophys Res Commun, 2003 Nov 21, 311(3), 604 - 9
Temperature-dependent stability and translation of Escherichia coli ompA mRNA; Afonyushkin T et al.; RNase E is known to affect the turnover of ompA mRNA in a growth rate-dependent manner . Here, we show that this enzyme also plays a role in the temperature-dependent stability of the transcript, thereby maintaining comparable levels of OmpA at 28 and 37 degrees C . An increase in the efficiency of RNase E cleavages at 37 degrees C within the 5(') UTR of the transcript in vitro was found to correlate with a decreased half-life and steady-state level at elevated temperature in vivo . However, measurements of de novo OmpA synthesis and in vitro toeprinting experiments suggest that translation of ompA mRNA is more efficient at 37 degrees C when compared to 28 degrees C . Thus, the enhanced translation apparently counteracts the decreased half-life at elevated temperature . Moreover, we propose that the temperature-dependent inverse correlation between ompA mRNA stability and translation can result from structural changes induced in the 5(') UTR of the transcript.

Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 847 - 52
Protoporphyrin IX binding and transport by recombinant mouse PBR; Wendler G et al.; The mitochondrial 18kDa peripheral-type benzodiazepine receptor (PBR), a high affinity cholesterol binding protein, has been shown to interact with protoporphyrin IX (PPIX) and this interaction was assumed to be involved in the regulation of heme biosynthesis and porphyrin-based photodynamic therapy in cancer . In order to test this hypothesis recombinant mouse PBR was expressed in Escherichia coli . The recombinant gene product showed in E . coli protoplasts specific affinity for PPIX binding . PPIX could displace PK 11195 binding . Moreover, induced PBR protein expression in E . coli protoplasts caused an uptake of PPIX that could be completely inhibited by cholesterol and to a lesser extent inhibited by PK 11195 and Ro5-4864 . These results suggest that PBR, in addition to its role in cholesterol and coproporphyrinogen III transport, is also directing the mitochondrial PPIX import, a function that can be ascribed to the 18kDa PBR protein alone.

Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 822 - 8
Bystander cell killing spreading from endothelial to tumor cells in a three-dimensional multicellular nodule model after Escherichia coli nitroreductase gene delivery; Benouchan M et al.; Tumor cells are elusive targets for standard anticancer chemotherapy due to their heterogeneity and genetic instability . On the other hand, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate . Thus, antiangiogenic therapy directed against tumor's ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance . Here we present a gene-directed enzyme prodrug therapy (GDEPT) strategy for targeting the tumor vasculature, using the Escherichia coli nitroreductase (ntr) gene delivery associated with the treatment with the prodrug CB1954 . In a first time we demonstrated the ability of the ntr/CB1954 system to induce an apoptotic-mediated cell death on monolayer cultures of human umbilical vein ECs (HUV-EC-C) . Then, when ntr-transfected HUV-EC-C cells (HUV-EC-C/ntr(+)) were associated in a three-dimensional (3-D) multicellular nodule model with untransfected B16-F10 murine melanoma cell line, we observed a CB1954-mediated bystander cell killing effect from endothelial to neighboring melanoma cells . To our knowledge, this is the first report indicating that GDEPT-based antiangiogenic targeting may be an effective approach for cancer treatment relied on the spreading of the bystander effect from endothelial to tumor cells.

J Mol Biol, 2003 Nov 28, 334(3), 595 - 603
Glucocorticoid receptor-like Zn(Cys)4 motifs in BslI restriction endonuclease; Vanamee ES et al.; BslI restriction endonuclease cleaves the symmetric sequence CCN(7)GG (where N=A, C, G or T) . The enzyme is composed of two subunits, alpha and beta, that form a heterotetramer (alpha(2)beta(2)) in solution . The alpha subunit is believed to be responsible for DNA recognition, while the beta subunit is thought to mediate cleavage . Here, for the first time, we provide experimental evidence that BslI binds Zn(II) . Specifically, using X-ray absorption spectroscopic analysis we show that the alpha subunit of BslI contains two Zn(Cys)(4)-type zinc motifs similar to those in the DNA-binding domain of the glucocorticoid receptor . This conclusion is supported by genetic analysis of the zinc-binding motifs, whereby amino acid substitutions in the zinc finger motifs are demonstrated to abolish or impair cleavage activity . An additional putative zinc-binding motif was identified in the beta subunit, consistent with the X-ray absorption data . These data were corroborated by proton induced X-ray emission measurements showing that full BslI contains at least three fully occupied Zn sites per alpha/beta heterodimer . On the basis of these data, we propose a role for the BslI Zn motifs in protein-DNA as well as protein-protein interactions.

J Mol Biol, 2003 Nov 28, 334(3), 527 - 39
Inter-molecular coiled-coil formation in human apolipoprotein E C-terminal domain; Choy N et al.; Human apolipoprotein E (apoE) is composed of an N-terminal (NT) domain (residues 1-191) that bears low-density lipoprotein receptor-binding sites, and a C-terminal (CT) domain (residues 210-299), which houses lipoprotein binding and apoE self-association sites . The NT domain is comprised of a four-helix bundle, while the structural organization of the CT domain is not known . Secondary structural algorithms predict that the apoE CT domain adopts an amphipathic alpha-helical conformation . On the basis of further sequence predictions, we identified a segment (residues 218-266) in the apoE CT domain that bears a high propensity to form a coiled-coil helix, which coincides with the putative lipoprotein-binding surface . An apoE construct bearing residues 201-299 that encompasses the entire CT domain was designed, expressed in Escherichia coli and purified by affinity chromatography . Circular dichroism (CD) spectroscopy of the apoE CT domain reveals spectra characteristic of coiled-coil helices, with the ratio of molar ellipticities at 222 nm and 208 nm ({theta}(222)/{theta}(208)) of 1.03 . Trifluoroethanol (TFE) stabilized the secondary structure of the apoE CT domain and disrupted coiled-coil helix formation as determined by CD and tryptophan fluorescence analysis . Analytical ultracentrifugation and lysine-specific cross-linking analysis of the apoE CT domain revealed predominant formation of dimeric and tetrameric species in aqueous buffers, and monomeric forms in 50% TFE . Guanidine hydrochloride-induced denaturation studies reveal that, at low concentrations of denaturant, the apoE CT domain maintains the {theta}(222)/{theta}(208) ratio at approximately 1.0 and elicits an altered tertiary environment with a shift in oligomeric state towards a dimer, indicative of the role of coiled-coil helix formation in inter molecular interactions . Further, coiled-coil formation is disrupted by protonation below pH 6.0, with a corresponding decrease in Trp fluorescence emission intensity, demonstrating that salt-bridge interactions play a critical role in maintaining the structural integrity of the apoE CT domain . The data support the concept that inter molecular coiled-coil helix formation is an essential structural feature of the apoE CT domain, which likely plays a role in clustering heparin-binding sites and/or sequestering the lipid-binding surface in lipid-free states.

J Mol Biol, 2003 Nov 28, 334(3), 489 - 99
Domain motions in GroEL upon binding of an oligopeptide; Wang J et al.; GroEL assists protein folding by preventing the interaction of partially folded molecules with other non-native proteins . It binds them, sequesters them, and then releases them so that they can fold in an ATP-driven cycle . Previous studies have also shown that protein substrates, GroES, and oligopeptides bind to partially overlapped sites on the apical domain surfaces of GroEL . In this study, we have determined the crystal structure at 3.0A resolution of a symmetric (GroEL-peptide)(14) complex . The binding of each of these small 12 amino acid residue peptides to GroEL involves interactions between three adjacent apical domains of GroEL . Each peptide interacts primarily with a single GroEL subunit . Residues R231 and R268 from adjacent subunits isolate each substrate-binding pocket, and prevent bound substrates from sliding into adjacent binding pockets . As a consequence of peptide binding, domains rotate and inter-domain interactions are greatly enhanced . The direction of rotation of the apical domain of each GroEL subunit is opposite to that of its intermediate domain . Viewed from outside, the apical domains rotate clockwise within one GroEL ring, while the ATP-induced apical domain rotation is counter-clockwise.

J Mol Biol, 2003 Nov 28, 334(3), 459 - 76
Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase; Marco-Marin C et al.; We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis . The mutations K8R and D162E decreased V({sustrate}= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups . R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG . Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate . These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template . The model has good stereochemistry and validation parameters . It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine . Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain . We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric . Further, it is inactive, as demanded if dimer formation is essential for activity . Models for AKIII architecture are proposed that account for these findings.

J Mol Biol, 2003 Nov 28, 334(3), 435 - 44
Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide biosynthetic pathway: insights into extender unit biosynthesis; Watanabe K et al.; Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506 . Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules . The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4% . FkbI has a similar fold to acyl-CoA dehydrogenases . Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate . This structural observation coincided the genetic experiment done by Carroll et al . J . Am . Chem . Soc., 124 (2002) 4176 . Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis . A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.

FEBS Lett, 2003 Nov 20, 554(3), 485 - 8
The importance of the Q motif in the ATPase activity of a viral helicase; Gallivan JP et al.; NS3 proteins of flaviviruses contain motifs which indicate that they possess protease and helicase activities . The helicases are members of the DExD/H box helicase superfamily and NS3 proteins from some flaviviruses have been shown to possess ATPase and helicase activities in vitro . The Q motif is a recently recognised cluster of nine amino acids common to most DExD/H box helicases which is proposed to regulate ATP binding and hydrolysis . In addition a conserved residue occurs 17 amino acids upstream of the Q motif ('+17') . We have analysed full-length and truncated NS3 proteins from Powassan virus (a tick-borne flavivirus) to investigate the role that the Q motif plays in the hydrolysis of ATP by a viral helicase . The Q motif appears to be essential for the activity of Powassan virus NS3 ATPase, however NS3 deletion mutants that contain the Q motif but lack the '+17' amino acid have ATPase activity albeit at a reduced level.

FEBS Lett, 2003 Nov 20, 554(3), 357 - 62
Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria; Imamura S et al.; The expression of group 2 sigma factors is characterized in a cyanobacterium Synechocystis sp . PCC 6803 grown in culture, changing light conditions (white, red and blue light, and darkness), or the presence of drugs (rifampicin, chloramphenicol, DCMU, and DBMIB), and the roles of these sigma factors are elucidated . The expression of dark/light-induced SigB/SigD was accelerated under opposite redox (oxidation/reduction) states in an electron transport chain of photosynthesis . Expression of the dark-induced lrtA and light-induced psbA2/3 transcript was significantly reduced in the sigB and sigD knockout strains, respectively . Abundant amounts of sigB transcript and protein were observed in the sigC knockout strain, implying that SigC represses SigB expression under light . These findings clearly showed that SigB/SigD with another group 2 sigma, SigC, contribute to transcription for a subset of dark/light-responsive genes in the cyanobacterium . A possible model for SigB/SigD is presented and the potential ability for promoter recognition is also discussed.

FEBS Lett, 2003 Nov 20, 554(3), 319 - 22
Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome; Wang L et al.; Mitochondrial deoxyguanosine kinase (dGK) catalyzes the initial phosphorylation of purine deoxynucleosides . Mutations in the dGK gene leading to deficiency in dGK activity is one of the causes of severe mitochondrial DNA depletion diseases . We used site-directed mutagenesis to introduce the clinically observed genetic alterations in the dGK gene and characterized the recombinant enzymes . The R142K enzyme had very low activity with deoxyguanosine and no activity with deoxyadenosine . The E227K mutant enzyme had unchanged K(m) values for all its substrates but very low V(max) values . C-terminal truncated dGK proteins were inactive . These results may help to define the role of dGK in mitochondrial DNA (mtDNA) precursor synthesis.

FEBS Lett, 2003 Nov 20, 554(3), 253 - 6
Phosphorylation by glycogen synthase kinase of inhibitor-2 does not change its structure in free state; Lin TH et al.; Inhibitor-2 (I2) is a thermostable protein that specifically binds to the catalytic subunit of protein phosphatase-1 (PP1), resulting in the formation of the inactive holoenzyme, ATP-Mg-dependent phosphatase . Phosphorylation of I2 at Thr-72 by glycogen synthase kinase-3 (GSK-3) results in activation of the phosphatase, suggesting that kinase action triggers conformational change in the complex . In this paper, we characterize the effect of GSK-3 phosphorylation on the structure of free state I2{1-172} by nuclear magnetic resonance and circular dichroism spectroscopy, and show that phosphorylation has no significant effect on its conformation . We conclude that the conformational changes of ATP-Mg-dependent phosphatase induced by GSK-3 phosphorylation must depend on the interactions between PP1 and I2.

Microbes Infect, 2003 Nov, 5(13), 1189 - 93
Putative roles of the CNF2 and CDTIII toxins in experimental infections with necrotoxigenic Escherichia coli type 2 (NTEC2) strains in calves; Van Bost S et al.; Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred . This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir . In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes . The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine . Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs . Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.

Arch Biochem Biophys, 2003 Dec 1, 420(1), 95 - 102
Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells; Taguchi G et al.; In higher plants, secondary metabolites are often converted to their glycoconjugates by glycosyltransferases (GTases) . We cloned a cDNA encoding GTase (NtGT2) from tobacco (Nicotiana tabacum L.) . The recombinant enzyme expressed in Escherichia coli (rNTGT2) showed glucosylation activity against several kinds of phenolic compounds, particularly the 7-hydroxyl group of flavonoids and 3-hydroxycoumarin . The K(m) values of kaempferol and 3-hydroxycoumarin with rNTGT2 are 6.5 microM and 23.6 microM, respectively . The deduced amino acid sequence of NTGT2 shows 60-70% identity to that of anthocyanin 5-O-glucosyltransferase (A5GT); rNTGT2 did not show activity against the anthocyanins tested . NtGT2 gene expression was induced by treating tobacco cells with plant hormones such as salicylic acid . We consider that NtGT2 gene might have evolved from the same ancestral gene as the A5GT genes to the stress-inducible GTases that react on several phenolic compounds.

Arch Biochem Biophys, 2003 Dec 1, 420(1), 35 - 9
Novel sterols synthesized via the CYP27A1 metabolic pathway; Pikuleva I et al.; A major biologic role of the ubiquitous mitochondrial P450 enzyme CYP27A1 is the generation of ligands such as 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, which regulate the expression of nuclear receptors that govern many aspects of cholesterol homeostasis . We now report that sterol intermediates in cholesterol synthesis, beginning with the initial post-cyclization sterol, lanosterol, continuing with zymosterol, and ending with desmosterol are also substrates for the enzyme . Using the human enzyme expressed in Escherichia coli, we characterized the retention times and major mass fragments of these novel metabolites . Although sequestration of the enzyme in the inner mitochondrial membrane and normal subcellular organization probably greatly restrict the proportion of these and other intermediates in cholesterol synthesis that undergo side chain oxidation, disruption of compartmentalization can bypass cholesterol as the end product and give rise to potent ligands that further modify gene expression.

Arch Biochem Biophys, 2003 Dec 1, 420(1), 18 - 34
Sterol methyltransferase2: purification, properties, and inhibition; Zhou W et al.; Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca . 40 kDa on SDS-PAGE and recognizes native sterols synthesized by Arabidopsis with a Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM and k(cat) 0.01 s(-1)) . Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-ethyl side chains . Structural determinants essential to activity were the nucleophilic features at C-3 and C-24 . The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Delta(24(24))<Delta(24(28)) and nucleus, Delta(7)<Delta(8)<Delta(5)=9,19-cyclopropane . The 14 alpha-methyl group was harmful to catalysis, reducing the suitability of cycloartenol as a substrate . On the basis of substrate activity and product distribution, SMT action was probed further using substrate (26,27-dehydrozymosterol: 26,27-DHZ) and intermediate (25-azacycloartenol: 25-AC) analogs of the SMT-catalyzed reactions . 26,27-DHZ was C-methylated to 26-homocholesta-8(9), 23(24)E, 26(26('))-trienol as well as 26-homocholesta-8(9),26(26')-3 beta,24 beta-dienol by SMT2, K(m) of 15 microM, k(cat) of 0.001 s(-1) . In addition, 26,27-DHZ acted as a mechanism-based irreversible inhibitor that results in the specific covalent modification of SMT2, exhibiting K(i) of 49 microM, k(inact) of 0.009 s(-1) and partition ratio of 0.11 . Substrate protection with zymosterol, 24(28)-methylenelophenol against 26,27-DHZ and similar inhibition of the first and second C(1)-transfer activities by the reversible inhibitor 25-AC of K(i) 20 nM suggested the analogs interacted at the same active site . {28E-2H}- and {28Z-2H}24(28)-methylenelanosterols were paired with AdoMet and differences of 2H-incorporation in the enzyme-generated 24-ethyl olefins supported an antimechanism . The results suggest plant SMT2 has a position-specific substrate specificity for Delta(24(25))-sterols and contains a single active center to catalyze the consecutive C(1)-transfer activities by substrate reaction channels similar to the fungal SMT1.

Cell, 2003 Nov 14, 115(4), 437 - 47
Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking; Neuman KC et al.; RNA polymerase (RNAP) transcribes DNA discontinuously, with periods of rapid nucleotide addition punctuated by frequent pauses . We investigated the mechanism of transcription by measuring the effect of both hindering and assisting forces on the translocation of single Escherichia coli transcription elongation complexes, using an optical trapping apparatus that allows for the detection of pauses as short as one second . We found that the vast majority of pauses are brief (1-6 s at 21 degrees C, 1 mM NTPs), and that the probability of pausing at any particular position on a DNA template is low and fairly constant . Neither the probability nor the duration of these ubiquitous pauses was affected by hindering or assisting loads, establishing that they do not result from the backtracking of RNAP along the DNA template . We propose instead that they are caused by a structural rearrangement within the enzyme.

Mol Microbiol, 2003 Nov, 50(4), 1381 - 90
The quaternary structure of RNase G from Escherichia coli; Briant DJ et al.; RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA . This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli . In this study, we developed non-denaturing purifications for overexpressed RNase G . Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites . Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers . Moreover, dimerization is required for activity . Four of the six cysteine residues of RNase G were mutated to serine . No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity . However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers . We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.

Mol Microbiol, 2003 Nov, 50(4), 1349 - 60
Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA; Cairrao F et al.; In this paper we show that RNase R is a cold shock protein that is induced seven- to eightfold by cold shock and that its expression is tightly regulated by temperature . Transcriptional studies reveal that the rnr gene is co-transcribed with flanking genes as an operon induced under cold shock . The induction of RNase R levels is mainly a result of the stabilization of the rnr transcripts . The transient stability of the rnr transcripts is shown to be regulated by PNPase at the end of the acclimation phase . Studies with an rnr mutant revealed a cold-shock phenotype showing that RNase R contributes to growth at low temperatures . We have shown that RNase R can be involved in the maturation of SsrA/tmRNA, an important small stable RNA involved in protein tagging and ribosome rescue . The wide biological significance of RNase R regarding adaptation to cold shock and its involvement in RNA surveillance, protein quality control and pathogenesis is discussed.

Mol Microbiol, 2003 Nov, 50(4), 1339 - 47
Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae; Bardy SL et al.; The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses . One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament . The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK . FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay . Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype . The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide . Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay . Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin . As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.

Mol Microbiol, 2003 Nov, 50(4), 1283 - 94
Inactivation of the decay pathway initiated at an internal site by RNase E promotes poly(A)-dependent degradation of the rpsO mRNA in Escherichia coli; Marujo PE et al.; In Escherichia coli, RNA degradation is mediated by endonucleolytic processes, frequently mediated by RNase E, and also by a poly(A)-dependent mechanism . The dominant pathway of decay of the rpsO transcripts is initiated by an RNase E cleavage occurring at a preferential site named M2 . We demonstrate that mutations which prevent this cleavage slow down degradation by RNase E . All these mutations reduce the single-stranded character of nucleotides surrounding the cleavage site . Moreover, we identify two other cleavage sites which probably account for the slow RNase E-mediated degradation of the mutated mRNAs . Failure to stabilize the rpsO transcript by appending a 5' hairpin indicates that RNase E is not recruited by the 5' end of mRNA . The fact that nucleotide substitutions which prevent cleavage at M2 facilitate the poly(A)-dependent degradation of the rpsO transcripts suggest an interplay between the two mechanisms of decay . In the discussion, we speculate that a structural feature located in the vicinity of M2 could be an internal degradosome entry site promoting both RNase E cleavages and poly(A)-dependent degradation of the rpsO mRNA . We also discuss the role of poly(A)-dependent decay in mRNA metabolism.

Mol Microbiol, 2003 Nov, 50(4), 1111 - 24
Global analysis of small RNA and mRNA targets of Hfq; Zhang A et al.; Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs . Hfq binds this family of small RNAs efficiently . We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family . This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected . At least 15 of 46 known small RNAs in E . coli interact with Hfq . In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis . Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets . Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation . Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.

Eur J Biochem, 2003 Nov, 270(21), 4356 - 64
Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli; Orhanovic S et al.; Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits {Orhanovic S., Pavela-Vrancic M . and Flogel-Mrsic M . (1994) Acta . Pharm.44, 87-95} . The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg2+ binding, has been examined . The influence of the metal ion content on the catalytic properties of APase from E . coli has been examined by kinetic analyses . An activation study has indicated that Mg2+ enhances APase activity by a mechanism that involves interactions between subunits . The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn2+ or Mg2+ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme . In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed . The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product . In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release . APase displays higher activity in the presence of Mg2+, as binding of Mg2+ increases the rate of conformational change . A conformationally controlled and Mg2+-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of Pi into the environment.

Eur J Biochem, 2003 Nov, 270(21), 4339 - 47
Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani; Villa H et al.; ATP-regenerating enzymes may have an important role in maintaining ATP levels in mitochondria-like kinetoplast organelle and glycosomes in parasitic protozoa . Adenylate kinase (AK) (ATP:AMP phosphotransferase) catalyses the reversible transfer of the gamma-phosphate group from ATP to AMP, releasing two molecules of ADP . This study describes cloning and functional characterization of the gene encoding AK2 from a genomic library of Leishmania donovani and also its expression in leishmania promastigote cultures . AK2 was localized on an approximately 1.9-Mb chromosomal band as a single copy gene . L . donovani AK2 gene is expressed as a single 1.9-kb mRNA transcript that is developmentally regulated and accumulated during the early log phase . The overexpression of L . donovani AKgene in Escherichia coli yielded a 26-kDa polypeptide that could be refolded to a functional protein with AK activity . The recombinant protein was purified to apparent homogeneity . Kinetic analysis of purified L . donovani AK showed hyperbolic behaviour for both ATP and AMP, with Km values of 104 and 74 microM, respectively . The maximum enzyme activity (Vmax) was 0.18 micromol.min(-1).mg(-1) protein . P1,P5-(bis adenosine)-5'-pentaphosphate (Ap5A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated Ki values of 190 nM and 160 nM for ATP and AMP, respectively . Ap5A also inhibited the growth of L . donovani promastigotes in vitro which could be only partially reversed by the addition of ADP . Thus, presence of a highly regulated AK2, which may have role in maintenance of ADP/ATP levels in L . donovani, has been demonstrated.

Eur J Biochem, 2003 Nov, 270(21), 4294 - 305
Functional effects of deleting the coiled-coil motif in Escherichia coli elongation factor Ts; Karring H et al.; Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome . The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal . In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement . The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used . The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain . Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu . However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex . The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu . The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.

Eur J Biochem, 2003 Nov, 270(21), 4272 - 81
Thioredoxin reductase from the malaria mosquito Anopheles gambiae; Bauer H et al.; The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria . Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A . gambiae and functionally substituted by the thioredoxin system . The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol . The A . gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant . The predominant isoform, A . gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells . In redox titrations, the substrate A . gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A . gambiae thioredoxin reductase-1 to the fully oxidized state . This indicates that, in contrast to other disulfide reductases, A . gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state . The thioredoxin reductases of the malaria system were compared . A . gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P . falciparum, respectively . A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation . The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A . gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P . falciparum enzyme . These differences offer an interesting approach to the design of species-specific inhibitors . Notably, A . gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.

Eur J Biochem, 2003 Dec, 270(23), 4771 - 9
Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli . Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials; Hirata S et al.; On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand . In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex . However, the Fe(III) complexes of these mutants are six-coordinated low-spin, like the wild-type enzyme . The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95 . To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein . CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme . The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were considerably lower than that of the wild-type enzyme (+70 mV) at -1, -26, and -122 mV vs . SHE, respectively . Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme.

Eur J Biochem, 2003 Dec, 270(23), 4671 - 80
Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis . A key factor participating in natural rubber biosynthesis; Asawatreratanakul K et al.; Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration . The enzyme responsible for the cis-1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants . By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H . brasiliensis for a functional factor participating in natural rubber biosynthesis . Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase . Northern blot analysis indicated that the transcript(s) of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined . In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 x 103-1 x 104 Da . Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased . These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber.

Biochemistry, 2003 Nov 25, 42(46), 13684 - 97
Mechanism of Cph1 phytochrome assembly from stopped-flow kinetics and circular dichroism; Borucki B et al.; The kinetics and mechanism of the autocatalytic assembly of holo-Cph1 phytochrome (from Synechocystis) from the apoprotein and the bilin chromophores phycocyanobilin (PCB) and phycoerythrobilin (PEB) were investigated by stopped flow and circular dichroism . At 1:1 stoichiometry, pH 7.9, and 10 degrees C, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants tau(1) approximately 150 ms, tau(2) approximately 2.5 s, and tau(3) approximately 50 s . Tau(1) was associated with a major red shift and transfer of oscillator strength from the Soret region to the 680 nm region . When the sulfhydryl group of cysteine 259 was blocked with iodoacetamide, preventing the formation of a covalent adduct, a noncovalent red-shifted complex (680 nm) was formed with a time constant of 200 ms . Tau(1) could thus be assigned to the formation of a noncovalent complex . The absorption changes during tau(1) are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket . From the concentration and pH dependence of the kinetics we obtained a value of 1.5 microM for the K(D) of this noncovalent complex and a value of 8.4 for the pK(a) of the proton donor . The tau(2) component was associated with a blue shift of about 25 nm and was attributed to the formation of the covalent bond (P(r)), accompanied with the loss of the 3-3' double bond to ring A . Tau(3) was due to photoconversion to P(fr) . For PEB, which is not photochromic, the formation of the noncovalent complex is faster (tau(1) = 70 ms), but the covalent bond formation is about 80 times slower (tau(2) = 200 s) than with the natural chromophore PCB . The CD spectra of the PCB adduct in the 250-800 nm range show that the chromophore geometries in P(r) and P(fr) are similar to those in plant phytochrome . The opposite rotational strengths of P(r) and P(fr) in the longest wavelength band suggest that the photoisomerization induces a reversal of the chirality . The Cph1 complex with noncovalently bound PCB was still photochromic when cysteine 259 was blocked with IAA or with the bulkier IAF . The covalent linkage to cysteine 259 is thus not required for photoconversion . The CD spectra of the noncovalently bound PCB in P(r)- and P(fr)-like states are qualitatively similar to those of the covalent adducts, suggesting analogous structures in the binding pocket . The noncovalent interactions with the binding pocket are apparently sufficient to hold the chromophore in the appropriate geometry for photoisomerization.

Biochemistry, 2003 Nov 25, 42(46), 13541 - 50
NMR structure of an archaeal homologue of ribonuclease P protein Rpp29; Sidote DJ et al.; A protein component of the Archaeoglobus fulgidus RNase P was expressed in Escherichia coli, purified, and structurally characterized using multidimensional NMR methods . The dominant structural feature of this 11 kDa protein is a sheet of six antiparallel beta-strands, wrapped around a core of conserved hydrophobic amino acids . Amide proton exchange and (15)N relaxation rate data provide evidence that the first 16 residues of the protein, located before the start of the first beta-strand, and the last 24 residues, located past the end of the last beta-strand, are relatively flexible; this contrasts with the relatively rigid and well-defined structure of the beta-sheet . Amino acid sequence comparisons among a diverse set of species indicate that the A . fulgidus protein is homologous to the human RNase P protein Rpp29, yeast RNase P protein Pop4, and a known archaeal RNase P protein from Methanobacter thermoautotrophicus; conserved hydrophobic residues indicate that the homologous protein in each of these species contains a similar beta-sheet structure . Conserved surface residues located in the loop connecting strands beta2 and beta3, the loop connecting strands beta4 and beta5, and in the flexible N- and C-terminal tails are most likely to have specific interactions with the RNA and other proteins of RNase P . The structural model of an RNase P protein component provided by the present work provides an essential step toward eventually understanding the overall architecture of this complex enzyme and the mechanism by which it performs its functions.

Biochemistry, 2003 Nov 25, 42(46), 13536 - 40
Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation; Ivanova MI et al.; Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA) . While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils . When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids . Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids . The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.

Biochemistry, 2003 Nov 25, 42(46), 13529 - 35
Ubiquitin recognition by the DNA repair protein hHR23a; Wang Q et al.; Ubiquitin is a prominent regulatory protein in numerous biological processes, including targeted protein degradation, endocytic sorting, transcriptional control, intranuclear localization, and retroviral virion budding . Ubiquitin-associated (UBA) domains, ubiquitin interacting motifs (UIM), and coupling of ubiquitin conjugation to ER degradation (CUE) motifs have been identified as ubiquitin receptors . The DNA repair protein hHR23a has two UBA domains that can each bind ubiquitin in addition to an N-terminal UBL domain that binds S5a and S2, two components of the 26S proteasome . Here we reveal hHR23a recognizes ubiquitin through a predominately hydrophobic surface formed by residues within alpha1 and alpha3 of each of its UBA domains . These two UBA surfaces bind a region on ubiquitin that includes K48 . These findings have implications for published studies revealing that hHR23a inhibits K48-linked polyubiquitin chain formation . In addition, by using (15)N NMR relaxation experiments, we find that binding ubiquitin requires a structural change in hHR23a . HHR23 proteins are hypothesized to link ubiquitin to S5a, and we provide direct evidence that hHR23 could form a ternary complex with ubiquitin and S5a.

Biochemistry, 2003 Nov 25, 42(46), 13522 - 8
Recombinant human cathepsin H lacking the mini chain is an endopeptidase; Vasiljeva O et al.; Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli . Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced . Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold) . Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H . However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration . Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14 . Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors . These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.

Biochemistry, 2003 Nov 25, 42(46), 13512 - 21
Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity; Zhang Y et al.; The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the alpha-Gal epitope . alpha3GT catalyzes galactose transfer from UDP-Gal to beta-linked galactosides, such as lactose, and in the absence of an acceptor substrate, to water at a lower rate . We have used site-directed mutagenesis to investigate the roles in catalysis and specificity of residues in alpha3GT that form H-bonds as well as other interactions with substrates . Mutation of the conserved Glu(317) to Gln weakens lactose binding and reduces the k(cat) for galactosyltransfer to lactose and water by 2400 and 120, respectively . The structure is not perturbed by this substitution, but the orientation of the bound lactose molecule is changed . The magnitude of these changes does not support a previous proposal that Glu(317) is the catalytic nucleophile in a double displacement mechanism and suggests it acts in acceptor substrate binding and in stabilizing a cationic transition state for cleavage of the bond between UDP and C1 of the galactose . Cleavage of this bond also linked to a conformational change in the C-terminal region of alpha3GT that is coupled with UDP binding . Mutagenesis indicates that His(280), which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal, is a key residue in the stringent donor substrate specificity through its role in stabilizing the bound UDP-Gal in a suitable conformation for catalysis . Mutation of Gln(247), which forms multiple interactions with acceptor substrates, to Glu reduces the catalytic rate of galactose transfer to lactose but not to water . This mutation is predicted to perturb the orientation or environment of the bound acceptor substrate . The results highlight the importance of H-bonds between enzyme and substrates in this glycosyltransferase, in arranging substrates in appropriate conformations and orientation for efficient catalysis . These factors are manifested in increases in catalytic rate rather than substrate affinity.

Biochemistry, 2003 Nov 25, 42(46), 13505 - 11
Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase; Juers DH et al.; The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics . Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation . Binding of the competitive inhibitor isopropyl thio-beta-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type . In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type . Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol . By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state . The result is that the rate constant for galactosylation is increased but degalactosylation is slower . The covalent intermediate may be better stabilized than the second transition state . The substitution also results in better binding of glucose to both the free and the galactosylated enzyme . However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme . This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.

Biochemistry, 2003 Nov 25, 42(46), 13496 - 504
Steady-state kinetics and molecular evolution of Escherichia coli MenD {(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase}, an anomalous thiamin diphosphate-dependent decarboxylase-carboligase; Bhasin M et al.; (1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide . The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others . However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate . Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction . The protein is a dimer and shows cooperativity with respect to both substrates . The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD . MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp . NRC-1 and the eukaryote Arabidopsis thaliana . Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP . A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis . There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.

Biochemistry, 2003 Nov 25, 42(46), 13429 - 37
Crystal structure of NusA from Thermotoga maritima and functional implication of the N-terminal domain; Shin DH et al.; We report the crystal structure of N-utilizing substance A protein (NusA) from Thermotoga maritima (TmNusA), a protein involved in transcriptional pausing, termination, and antitermination . TmNusA has an elongated rod-shaped structure consisting of an N-terminal domain (NTD, residues 1-132) and three RNA binding domains (RBD) . The NTD consists of two subdomains, the globular head and the helical body domains, that comprise a unique three-dimensional structure that may be important for interacting with RNA polymerase . The globular head domain possesses a high content of negatively charged residues that may interact with the positively charged flaplike domain of RNA polymerase . The helical body domain is composed of a three-helix bundle that forms a hydrophobic core with the aid of two neighboring beta-strands . This domain shows structural similarity with one of the helical domains of sigma(70) factor from Escherichia coli . One side of the molecular surface shows positive electrostatic potential suitable for nonspecific RNA interaction . The RBD is composed of one S1 domain and two K-homology (KH) domains forming an elongated RNA binding surface . Structural comparison between TmNusA and Mycobacterium tuberculosis NusA reveals a possible hinge motion between NTD and RBD . In addition, a functional implication of the NTD in its interaction with RNA polymerase is discussed.

Biochemistry, 2003 Nov 25, 42(46), 13379 - 85
Template-directed assembly of receptor signaling complexes; Shrout AL et al.; Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity . The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar . Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup . In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF . The extent of CheA activation was found to be independent of the level of covalent modification on the CF . Instead, the stability of the complex increased significantly as the level of covalent modification increased . Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR . Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.

Genet Res, 2003 Aug, 82(1), 33 - 40
Long and short mRnas transcribed from the medaka fish transposon Tol2 respectively exert positive and negative effects on excision; Tsutsumi M et al.; The medaka fish transposable element, Tol2, is a member of the hAT family of transposons . It has been directly demonstrated to be active and two mRNAs, differing in length, have been isolated . They cover exons 1-4 and exons 2-4 and the longer form has already been proven to catalyse transposition reactions . However, the function of the shorter mRNA in medaka cells has hitherto remained unclear . In the present study, first we constructed a quantitative system to detect Tol2 excision using an indicator plasmid carrying a non-autonomous Tol2 within its lacZ gene; second we injected mRNAs with the plasmid into medaka eggs . Excision of Tol2 was detected as E . coli blue colonies caused by the recovery of lacZ activity . Addition of the longer mRNA increased excision, but the shorter did not . Moreover, co-injection of both mRNAs greatly lowered the frequency compared with the case of treatment with the longer mRNA alone . These results indicate that the shorter mRNA has an inhibitory effect on the excision reaction, and that the N-terminal region of the transposase encoded by exon 1, including a BED zinc finger, presumably plays an important role in excision . Here, we suggest a regulatory mechanism of Tol2 transposition involving the expression of these mRNAs.

Environ Sci Technol, 2003 Nov 1, 37(21), 5015 - 20
Ultraviolet disinfection: similitude in Taylor-Couette and channel flow; Forney LJ et al.; The inactivation data for Escherichia coli are recorded for the three reactor geometries of Taylor-Couette flow and flow between either concentric cylinders or a square channel . All of the data are shown to be correlated with the assumption of plug flow . In particular, the effects of nonuniform radiation levels are accounted for by integration across the fluid channel as done previously . However, a new correction factor is introduced that is shown to be inversely proportional to the laminar, velocity boundary thickness to account for the effects of a concentration boundary layer of surviving pathogen . It has also been demonstrated that the common problems of nonuniform radiation levels and concentration boundary layer effects in UV reactors are largely eliminated with the use of Taylor-Couette flow . Moreover, the repetitive exposure of fluid parcels to a small number of lamps in the rotating Taylor-Couette flow decreases maintainance requirements compared to the hydrodynamic equivalent of cross-flow over a tube bank or lamp array . Over a 3-log reduction in the inactivation of E . coli was demonstrated compared to a conventional channel with the same radiation dosage . Moreover, greater than a 2-log reduction was evident compared to flow through concentric cylinders.

Environ Sci Technol, 2003 Nov 1, 37(21), 4962 - 70
Evaluation of bioanalytical assays for toxicity assessment and mode of toxic action classification of reactive chemicals; Harder A et al.; The toxicity of electrqphiles, including reactive organochlorines, epoxides, and compounds with an activated double bond was investigated . A set of different bioanalytical assays based on genetically modified Escherichia coli strains was set up to quantify cytotoxicity and specific reactivity toward the important biological nucleophiles DNA and glutathione (GSH) . The significance of GSH for detoxification was assessed by cellular GSH depletion as well as by growth inhibition of a GSH-deficient strain . Tests for DNA damage comprised the measurement of induction of DNA repair systems, DNA fragmentation, and growth inhibition of a strain deficient in major DNA repair mechanisms . The most suitable assays for detection of mechanisms that underlie the observable cytotoxicity of the tested electrophiles were two sets of strains either lacking GSH or DNA repair in combination with their corresponding parent strains . Comparison of toxicity observed in those strains suggests three clearly distinguishable modes of toxic action for electrophilic chemicals: "DNA damage", "GSH depletion-related toxicity", and "unspecific reactivity" . The class of chemicals causing DNA damage includes the epoxides 1,2-epoxybutane, (2,3-epoxypropyl)benzene, and styrene oxide . The class of chemicals with GSH depletion-related toxicity includes compounds with an activated double bond, like acrylates and acrolein . All reactive organochlorines and some epoxides were classified as unspecifically reactive because their toxicity is initiated by reactions with both biological nucleophiles . The work presented here is a contribution for an alternative hazard and effect assessment of organic pollutants based on mode of toxic action classification.

Environ Sci Technol, 2003 Nov 1, 37(21), 4955 - 61
Applicability and limitation of OSARs for the toxicity of electrophilic chemicals; Harder A et al.; The appropriate selection and application of quantitative structure-activity relationships (QSARs) for the prediction of toxicity is based on the prior assignment of a chemical to its mode of toxic action . This classification is often derived from structural characteristics with the underlying assumption that chemically similar compounds have similar mechanisms of action, which is often but not necessarily the case . Instead of using structural characteristics for classification toward a mode of toxic action, we used Escherichia coli based bioanalytical assays to classify electrophilic chemicals . Analyzing a series of reactive organochlorines, epoxides, and compounds with an activated double bond, three subclasses of reactive toxicity were distinguished: "glutathione depletion-related toxicity", "DNA damage", and "unspecific reactivity" . For both subsets of specifically reacting compounds a direct correlation between effects and chemical reactivity was found . Reaction rate constants with either glutathione or 2'-deoxyguanosine, which was used as a model for complex DNA, served well to set up preliminary QSARs for either glutathione depletion-related toxicity or toxicity based on DNA damage in the model organism E . coli . The applicability of QSARs for electrophilic chemicals based on mechanistically relevant reaction rate constants is a priori limited to a small subset of compounds with strictly identical mechanism of toxic action and similar metabolic rates . In contrast, the proposed bioanalytical assays not only allowed the experimental identification of molecular mechanisms underlying the observable toxicity but also their toxicity values are applicable to quantitatively predict toxic effects in higher organisms by linear correlation models, independent of the assigned mode of toxic action.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Oct, 34(4), 610 - 3
{A study on the construction of allelic ladders for 4 short tandem repeat loci (D7S817, D10S1432, D10S1213 and D18S865) and their genetic polymorphism in two Chinese populations}; Jia Y et al.; OBJECTIVE: To produce standard allelic ladders for four STR loci(D7S817, D10S1432, D10S1213 and D18S865) by recombined plasmids and use them in the research of genetic polymorphism . METHODS: After being amplified by PCR, different STR allelic fragments were isolated from the polyacryramide gel . The STR allelic fragments were eluted into distilled water and reamplified by PCR to obtain purified allelic fragments . Next, the purified allelic fragments were subcloned individually into the PUC plasmid vectors, and the size and structure of the inserts were confirmed by the analysis of their DNA sequences . Then we transfected the PUC plasmid vectors into E . coli DH5 alpha cells, worked on selective culture, and finally, mixed the alleles of the four STR loci respectively . RESULTS: The standard allelic ladders for four new loci (D7S817, D10S1432, D10S1213 and D18S865) were obtained, with which the genetic polymorphisms of four new loci in Chinese Hans and Dongxiang ethnic groups were studied . CONCLUSION: The standard STR allelic ladders acquired by this method are of high value in forensic practice, and they are applicable to the analysis of genetic polymorphism.

Mol Genet Genomics, 2004 Jan, 270(6), 533 - 8 Epub 2003 Nov 14.
A comparative study of variation in codon 33 of the rpoS gene in Escherichia coli K12 stocks: implications for the synthesis of sigma(s); Subbarayan PR et al.; The Escherichia coli rpoS gene encodes an RNA polymerase sigma factor (sigma S or sigma(S)) required for the expression of stationary-phase genes . In the first published rpoS sequence from E . coli K-12 codon 33 is given as CAG . However, several subsequent independent studies found the amber codon TAG at this position ( rpoSAm) . Besides this amber codon, other codons such as TAT have also been found at this location in rpoS . Comparative genome analysis now leads us to propose TAG as the parental codon 33 in rpoS in E . coli K-12 . Five different stocks of the strain W3110, which differ in the levels of sigma(S) protein they express, were investigated . We sequenced the rpoS gene from these, and found a T at nucleotide position 97 in four out of the five stocks and a G at position 99 in three out of the five . W1485, a parental strain of W3110, and W3350, a derivative of W3110, are also rpoSAm mutants . Such rpoSAm mutants would be expected to show no RpoS activity . The retention of partial or intermediate sigma(S) activity by suppressor-free rpoSAm mutants is therefore puzzling . We propose that a functional, N-terminally truncated, sigma(S) (Delta1-53sigma(S)) can be translated from a Secondary Translation Initiation Region (STIR) located downstream of the amber codon 33 . It has recently been reported that a fragment of RpoS (Delta1-53sigma(S)) that lacks the first 53 amino acids is functional when synthesized in vivo . Taken together, our results support the hypothesis that the original codon 33 of the rpoS gene in E . coli K-12 strains is the amber codon TAG.

Virus Genes, 2003 Dec, 27(3), 257 - 62
ORF 5 of grapevine virus A encodes a nucleic acid-binding protein and affects pathogenesis; Galiakparov N et al.; A previous functional analysis of the genome of grapevine virus A (GVA) was not conclusive as to the role of open reading frame 5 (ORF 5) . This ORF encodes a 10-kDa protein (p10) carrying two distinct domains: a basic, arginine-rich domain and a zinc-finger domain . P10 was cloned and expressed in Escherichia coli, and was shown by northwestern assays to interact with nucleic acids . In-frame deletion of the basic region abolished P10's nucleic acid-binding capability, whereas substitution of cysteine residues by serine in the zinc-finger domain did not affect binding . These mutations were inserted into the full-length infectious clone . It has been shown that ORF 5 mutations do not affect replication of GVA-RNA . However, plants inoculated with the aforementioned mutations did not develop symptoms, and Western blot analysis revealed markedly reduced expression of the movement protein (the product of ORF 3).

J Bacteriol, 2003 Dec, 185(23), 7015 - 8
FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli; Giladi M et al.; Escherichia coli (thyA DeltafolA) mutants are viable and can grow in minimal medium when supplemented with thymidine alone . Here we present evidence from in vivo and in vitro studies that the ydgB gene determines an alternative dihydrofolate reductase that is related to the trypanosomatid pteridine reductases . We propose to rename this gene folM.

J Bacteriol, 2003 Dec, 185(23), 7001 - 7
The quorum sensing negative regulators EsaR and ExpR(Ecc), homologues within the LuxR family, retain the ability to function as activators of transcription; von Bodman SB et al.; Most LuxR homologues function as activators of transcription during the process of quorum sensing, but a few, including EsaR and ExpR(Ecc), negatively impact gene expression . The LuxR-activated luxI promoter and LuxR binding site, the lux box, were used in artificial contexts to assess the potential for transcriptional activation and DNA binding by EsaR and ExpR(Ecc) . Although the acyl-homoserine lactone responsiveness of both proteins is the opposite of that shown by most LuxR family members, EsaR and ExpR(Ecc) have preserved the ability to interact with RNA polymerase and activate transcription despite their low affinity for the lux box DNA.

J Bacteriol, 2003 Dec, 185(23), 6852 - 9
pH-Dependent modulation of cyclic AMP levels and GadW-dependent repression of RpoS affect synthesis of the GadX regulator and Escherichia coli acid resistance; Ma Z et al.; Extreme acid resistance is a remarkable property of virulent and avirulent Escherichia coli . The ability to resist environments in which the pH is 2.5 and below is predicted to contribute significantly to the survival of E . coli during passage through the gastric acid barrier . One acid resistance system imports glutamate from acidic environments and uses it as a proton sink during an intracellular decarboxylation reaction . Transcription of the genes encoding the glutamate decarboxylases and the substrate-product antiporter required for this system is induced under a variety of conditions, including the stationary phase and a low pH . Acid induction during log-phase growth in minimal medium appears to occur through multiple pathways . We recently demonstrated that GadE, the essential activator of the genes, was itself acid induced . In this report we present evidence that there is a regulatory loop involving cross-repression of two AraC-like regulators, GadX and GadW, that can either assist or interfere with GadE activation of the gad decarboxylase and antiporter genes, depending on the culture conditions . Balancing cross-repression appears to be dependent on cAMP and the cAMP regulator protein (CRP) . The control loop involves the GadX protein repressing the expression of gadW and the GadW protein repressing or inhibiting RpoS, which is the alternative sigma factor that drives transcription of gadX . CRP and cAMP appear to influence GadX-GadW cross-repression from outside the loop by inhibiting production of RpoS . We found that GadW represses the decarboxylase genes in minimal medium and that growth under acidic conditions lowers the intracellular cAMP levels . These results indicate that CRP and cAMP can mediate pH control over gadX expression and, indirectly, expression of the decarboxylase genes . Mutational or physiological lowering of cAMP levels increases the level of RpoS and thereby increases the production of GadX . Higher GadX levels, in turn, repress gadW and contribute to induction of the gad decarboxylase genes . The presence of multiple pH control pathways governing expression of this acid resistance system is thought to reflect different environmental routes to a low pH.

J Bacteriol, 2003 Dec, 185(23), 6747 - 55
Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli; Gauthier A et al.; Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones . It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus . The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system . We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC) . Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN . We showed by affinity chromatography that EscN and Tir bind CesT specifically . Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT . Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC . These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.

Am J Physiol Lung Cell Mol Physiol, 2004 Mar, 286(3), L573 - 9 Epub 2003 Nov 14.
Initial responses to ventilation of premature lambs exposed to intra-amniotic endotoxin 4 days before delivery; Ikegami M et al.; Preterm delivery is frequently preceded by chorioamnionitis, resulting in exposure of the fetal lung to inflammation . We hypothesized that ventilation of the antenatally inflamed lung would result in amplification of the lung injury . Therefore, we induced fetal lung inflammation with intra-amniotic endotoxin (10 mg of Escherichia coli 055:B5) 4 days before premature delivery at 130 days of gestation . Lung function and lung inflammation after surfactant treatment and 4 h of mechanical ventilation were evaluated . Inflammatory cell numbers in amniotic fluid were increased >10-fold by antenatal endotoxin exposure . Antenatal endotoxin exposure had minimal effects on blood pressure, heart rate, lung compliance, and blood gas values . The endotoxin-exposed lungs required higher ventilation pressures . Ventilation did not increase the number of inflammatory cells or the protein in bronchoalveolar lavage fluid of the endotoxin-exposed animals above that measured in endotoxin-exposed fetuses that were not ventilated . IL-1beta, IL-6, and IL-8 mRNA in cells from bronchoalveolar lavage fluid were increased by antenatal endotoxin exposure but not changed by ventilation . IL-1beta and IL-8 protein was increased in lung tissue by 4 h of ventilation . Very little inflammation was induced by ventilation in this premature lamb model of surfactant treatment and gentle ventilation . After lung inflammation was induced by intra-amniotic endotoxin injection, ventilation did not increase lung injury.

Mol Microbiol, 2003 Nov, 50(3), 961 - 75
The first committed step in the biosynthesis of sialic acid by Escherichia coli K1 does not involve a phosphorylated N-acetylmannosamine intermediate; Ringenberg MA et al.; A variety of pathogens or commensals use at least one of four distinct mechanisms for decorating their surfaces with sialic acid as a strategy to avoid, subvert or inhibit host innate immunity . The metabolism of sialic acid thus is central to a range of host-pathogen interactions . The first committed step in this process, the production of free N-acetylmannosamine (ManNAc), has not been defined . Here we show that ManNAc-6-phosphate (ManNAc-6-P) is not an obligate sialate precursor in Escherichia coli K1 . This conclusion was supported by 31P NMR spectroscopy of E . coli K1 derivatives engineered with different combinations of mutations in nanA (sialate aldolase or lyase), nanK (ManNAc kinase), nanE (ManNAc-6-P 2-epimerase), neuS (polysialyltransferase) and neuB (sialate synthase) . The product specificities for purified NanK and NanE were determined by chromatographic analyses . Direct biochemical analysis showed that ManNAc-6-P was stable in a nanE mutant extract . The combined results indicate that neither ManNAc-6-P nor specific or non-specific phosphatase are necessary to generate the requisite ManNAc for sialate biosynthesis . Our results imply that the neuC gene product encodes an UDP-N-acetylglucosamine 2-epimerase that generates ManNAc directly from the dinucleotide-sugar precursor despite detection of only this enzyme's UDP-GlcNAc hydrolase activity . This study describes the first use of NMR for analysing intermediate flux within the sialate biosynthetic pathway.

Mol Microbiol, 2003 Nov, 50(3), 897 - 909
Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli; Vecerek B et al.; The Escherichia coli Sm-like host factor I (Hfq) is thought to play direct and indirect roles in post-transcriptional regulation by targeting small regulatory RNAs and mRNAs . In this study, we have used proteomics to identify new mRNA targets of Hfq . We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq- background . The effect of Hfq on some of the corresponding mRNAs including fur, gapA, metF, ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods . This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs . From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq . Fur is known to be a negative regulator of transcription of the small RNA RyhB . Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur . These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.

Mol Microbiol, 2003 Nov, 50(3), 825 - 34
Segregation of the Escherichia coli chromosome terminus; Li Y et al.; We studied the segregation of the replication terminus of the Escherichia coli chromosome by time-lapse and still photomicroscopy . The replicated termini lie together at the cell centre . They rapidly segregate away from each other immediately before cell division . At fast growth rate, the copies move progressively and quickly toward the centres of the new-born cells . At slow growth rate, the termini usually remain near the inner cell pole and migrate to the cell centre in the middle of the cell cycle . A terminus domain of about 160kb, roughly centred on the dif recombination site, segregated as a unit at cell division . Sequences outside this domain segregated before division, giving two separate foci in predivision cells . Resolution of chromosome dimers via the terminus dif site requires the XerC recombinase and an activity of the FtsK protein that is thought to align the dif sequences at the cell centre . We found that anchoring of the termini at the cell centre and proper segregation at cell division occurred normally in the absence of recombination via the XerC recombinase . Anchoring and proper segregation were, however, frequently disrupted when the C-terminal domain of FtsK was truncated.

Plant J, 2003 Dec, 36(5), 664 - 74
Citrus fruit flavor and aroma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene; Sharon-Asa L et al.; Citrus fruits possess unique aromas rarely found in other fruit species . While fruit flavor is composed of complex combinations of soluble and volatile compounds, several low-abundance sesquiterpenes, such as valencene, nootkatone, alpha-sinensal, and beta-sinensal, stand out in citrus as important flavor and aroma compounds . The profile of terpenoid volatiles in various citrus species and their importance as aroma compounds have been studied in detail, but much is still lacking in our understanding of the physiological, biochemical, and genetic regulation of their production . Here, we report on the isolation, functional expression, and developmental regulation of Cstps1, a sesquiterpene synthase-encoding gene, involved in citrus aroma formation . The recombinant enzyme encoded by Cstps1 was shown to convert farnesyl diphosphate to a single sesquiterpene product identified as valencene by gas chromatography-mass spectrometry (GC-MS) . Phylogenetic analysis of plant terpene synthase genes localized Cstps1 to the group of angiosperm sesquiterpene synthases . Within this group, Cstps1 belongs to a subgroup of citrus sesquiterpene synthases . Cstps1 was found to be developmentally regulated: transcript was found to accumulate only towards fruit maturation, corresponding well with the timing of valencene accumulation in fruit . Although citrus fruits are non-climacteric, valencene accumulation and Cstps1 expression were found to be responsive to ethylene, providing further evidence for the role of ethylene in the final stages of citrus fruit ripening . Isolation of the gene encoding valencene synthase provides a tool for an in-depth study of the regulation of aroma compound biosynthesis in citrus and for metabolic engineering for fruit flavor characteristics.

Plant J, 2003 Dec, 36(5), 643 - 51
Regulation of Arabidopsis SHY2/IAA3 protein turnover; Tian Q et al.; Auxin/indole acetic acid (Aux/IAA) proteins regulate transcriptional responses to the plant hormone auxin . Gain-of-function mutations in the Arabidopsis SHORT HYPOCOTYL 2 (SHY2/IAA3) gene encoding an Aux/IAA protein increase steady-state levels of SHY2/IAA3 protein and decrease auxin responses, indicating that SHY2/IAA3 negatively regulates auxin signaling . These shy2 mutations also cause ectopic light responses, suggesting that SHY2/IAA3 may promote light signaling . Auxin regulates turnover of the related Auxin-resistant (AXR)2/IAA7 and AXR3/IAA17 proteins by increasing their interaction with the Skp1-Cdc53/cullin-F-box (SCFTIR1) E3 ubiquitin ligase complex . To investigate whether SHY2/IAA3 is regulated similarly, we have used a turnover assay to reveal that axr1 and transport inhibitor resistant (tir)1 mutations affecting SCFTIR1 decrease SHY2/IAA3 turnover . In pull-down assays, SHY2/IAA3 protein interacted with TIR1, the F-box component of SCFTIR1 and with the photoreceptor phytochrome B . Auxin stimulated SHY2/IAA3 interaction with TIR1, whereas the shy2-2 gain-of-function mutation decreased this interaction . Light did not affect the interaction, suggesting that light regulates some other aspect of Aux/IAA gene or protein function . The chemical juglone (5-hydroxy-1,4-naphthoquinone) inhibited the interaction, suggesting that peptidyl-prolyl isomerization may mediate auxin-induced SHY2/IAA3 protein turnover.

Plant J, 2003 Dec, 36(5), 616 - 28
Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts; Moberg P et al.; We have recently isolated and identified a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences . PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif . To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli . We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate specificity and intracellular localization . Mutagenesis studies of the residues involved in metal binding identified the histidines and the proximal glutamate as essential residues for the proteolytic activity . Substrate specificity studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides . The Zn-MP does not recognize an amino acid sequence per se . Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles . Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro . In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria--degradation of targeting peptides.

Clin Microbiol Infect, 2003 Aug, 9(8), 823 - 31
Comparison of PCR-based methods for typing Escherichia coli; Jonas D et al.; OBJECTIVE: To establish a library typing system appropriate for studying cross-transmission of Escherichia coli . METHODS: Eighteen epidemiologically unrelated isolates were genotyped by means of pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), repetitive (rep) PCR, and fluorescent amplified fragment length polymorphism (AFLP) . Fingerprints were analyzed either by Pearson correlation or, in the case of AFLP, by Dice coefficients employing the novel 'uncertain band' software tool from GelCompar II . During a nine-month period, 112 isolates taken from 93 patients hospitalized in five intensive care units were analyzed by use of the two most discriminative PCR typing methods . RESULTS: Genotyping by RAPD and rep-PCR revealed insufficient discrimination . Among 18 epidemiologically unrelated strains with 17 different PFGE patterns, IS3 rep-PCR and AFLP distinguished 10 and 18 types, respectively . Comparison of the different methods for analysis of AFLP fingerprints showed that the Dice coefficients, which ignore 'uncertain bands', offered the best concordance with visual interpretation . Consecutive isolates originating from the same patient differed in less than three fragments . CONCLUSIONS: AFLP analysis showed the highest discriminative capacity for PCR typing of E . coli isolates . Analysis of fingerprints employing the Dice coefficients proved the most efficient method for an automated software-based retrieval of visually indistinguishable genotypes in an AFLP fingerprint database.

Biochemistry (Mosc), 2003 Oct, 68(10), 1089 - 96
Effect of membrane phospholipid composition and charge of the signal peptide of Escherichia coli alkaline phosphatase on efficiency of its secretion; Golovastov VV et al.; The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(-20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase--protein SecA . Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion . In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases . The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein . If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity . The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion.

Annu Rev Genet, 2003, 37, 611 - 46
RecA-dependent recovery of arrested DNA replication forks; Courcelle J et al.; DNA damage encountered during the cellular process of chromosomal replication can disrupt the replication machinery and result in mutagenesis or lethality . The RecA protein of Escherichia coli is essential for survival in this situation: It maintains the integrity of the arrested replication fork and signals the upregulation of over 40 gene products, of which most are required to restore the genomic template and to facilitate the resumption of processive replication . Although RecA was originally discovered as a gene product that was required to change the genetic information during sexual cell cycles, over three decades of research have revealed that it is also the key enzyme required to maintain the genetic information when DNA damage is encountered during replication in asexual cell cycles . In this review, we examine the significant experimental approaches that have led to our current understanding of the RecA-mediated processes that restore replication following encounters with DNA damage.

Annu Rev Genet, 2003, 37, 31 - 66
Error-prone DNA polymerases: when making a mistake is the only way to get ahead; Rattray AJ et al.; Cells have high-fidelity polymerases whose task is to accurately replicate the genome, and low-fidelity polymerases with specialized functions . Although some of these low-fidelity polymerases are exceptional in their ability to replicate damaged DNA and restore the undamaged sequence, they are error prone on undamaged DNA . In fact, these error-prone polymerases are sometimes used in circumstances where the capacity to make errors has a selective advantage . The mutagenic potential of the error-prone polymerases requires that their expression, activity, and access to undamaged DNA templates be regulated . Here we review these specialized polymerases with an emphasis on their biological roles.

Plant Cell, 2003 Dec, 15(12), 3020 - 32 Epub 2003 Nov 13.
Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins; Wang H et al.; AL2 and L2 are related proteins encoded by geminiviruses of the Begomovirus and Curtovirus genera, respectively . Both are pathogenicity determinants that cause enhanced susceptibility when expressed in transgenic plants . To understand how geminiviruses defeat host mechanisms that limit infectivity, we searched for cellular proteins that interact with AL2 and L2 . Here, we present evidence that the viral proteins interact with and inactivate adenosine kinase (ADK), a nucleoside kinase that catalyzes the salvage synthesis of 5'-AMP from adenosine and ATP . We show that the AL2 and L2 proteins inactivate ADK in vitro and after coexpression in Escherichia coli and yeast . We also demonstrate that ADK activity is reduced in transgenic plants expressing the viral proteins and in geminivirus-infected plant tissues . By contrast, ADK activity is increased after inoculation of plants with diverse RNA viruses or a geminivirus lacking a functional L2 gene . Consistent with its ability to interact with multiple cellular kinases, we also demonstrate that AL2 is present in both the nucleus and the cytoplasm of infected plant cells . These data indicate that ADK is targeted by viral pathogens and provide evidence that this "housekeeping" enzyme might be a part of host defense responses . In previous work, we showed that AL2 and L2 also interact with and inactivate SNF1 kinase, a global regulator of metabolism that is activated by 5'-AMP . Together, these observations suggest that metabolic alterations mediated by SNF1 are an important component of innate antiviral defenses and that the inactivation of ADK and SNF1 by the geminivirus proteins represents a dual strategy to counter this defense . AL2 proteins also have been shown to act as suppressors of RNA silencing, an adaptive host defense response . A possible relationship between ADK inactivation and silencing suppression is discussed.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14086 - 90 Epub 2003 Nov 13.
On the relation between fluctuation and response in biological systems; Sato K et al.; A general relationship between fluctuation and response in a biological system is presented . The fluctuation is given by the variance of some quantity, whereas the response is given as the average change of that quantity for a given parameter change . We propose a relationship where the two are proportional, in a similar way to the fluctuation-dissipation theorem in physics . By studying an evolution experiment where fluorescence of protein in bacteria increases, we confirm our relation by observing a positive correlation between the speed of fluorescence evolution and the phenotypic fluctuation of the fluorescence over clone bacteria . The generality of the relationship as well as its relevance to evolution is discussed.

J Biol Chem, 2004 Feb 13, 279(7), 5072 - 80 Epub 2003 Nov 13.
Cox17 is functional when tethered to the mitochondrial inner membrane; Maxfield AB et al.; Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion . Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex . To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2 . The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17 . The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion . The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells . These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space . Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding . The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein . This motif can be spatially separated from the N-terminal copper-binding functional motif . Possible roles of the C-terminal motif are discussed.

Lab Invest, 2003 Nov, 83(11), 1657 - 67
Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells; Harada K et al.; To clarify the innate immunity of the intrahepatic biliary tree, we examined expression of Toll-like receptors and intracellular signalings in biliary epithelial cells in response to bacterial components by using cultured biliary epithelial cells (murine biliary cells and human cholangiocarcinoma cell lines) . The expression of Toll-like receptors in cultured cells was examined by reverse transcription and PCR and immunohistochemistry . Intracellular signalings after Toll-like receptors activation by lipopolysaccharide was examined by analysis of nuclear factor (NF)-kappaB activation and inhibition studies using inhibitors for NF-kappaB and mitogen-activated protein kinase and blocking antibody . The mRNAs of Toll-like receptors 2, 3, 4, and 5, and related molecules (MD-2, MyD88, and CD14) were detected, and their proteins were expressed in cultured cells . Lipopolysaccharide was shown to bind to the cell surface of cultured cells . Lipopolysaccharide treatment induced the production of TNF-alpha, and nuclear translocation of NF-kappaB and increased NF-kappaB-DNA binding in cultured cells . This induction of TNF-alpha was partially inhibited by anti-Toll-like receptor 4 antibody . The nuclear translocation and increased binding of NF-kappaB by lipopolysaccharide were blocked by addition of MG132, an inhibitor of NF-kappaB . In conclusion, lipopolysaccharide appears to form a receptor complex of CD14, Toll-like receptor 4, MD-2, and MyD88 in cultured biliary epithelial cells and seems to regulate activation of NF-kappaB and synthesis of TNF-alpha . The recognition of pathogen-associated molecular patterns using Toll-like receptors and related molecules in biliary epithelial cells, which is demonstrated in this in vitro study, may participate in an immunopathology of the intrahepatic biliary tree in vivo.

Vaccine, 2003 Dec 12, 22(2), 168 - 76
The immunogenicity, protective efficacy and safety of BBG2Na, a subunit respiratory syncytial virus (RSV) vaccine candidate, against RSV-B; Power UF et al.; Respiratory syncytial virus (RSV) is divided into subgroups A and B, based primarily on variation within the G glycoprotein . A safe vaccine that protects against both would be the ideal . BBG2Na is a recombinant subunit RSV vaccine candidate derived in part from the G protein of RSV-A . Interestingly, BBG2Na formulated in alum protected against RSV-B challenge at early time points following vaccination in mice . Over 6 months, however, BBG2Na-induced immunogenicity and protective efficacy progressively diminished, such that few animals were considered protected at the end . To study the safety of BBG2Na relative to RSV-B challenge, we established a novel enhanced immunopathology mouse model . We confirmed that RSV-B challenge of formalin-inactivated RSV-A (FI-RSV-A)-immunized BALB/c mice results in enhanced pulmonary pathology . Therefore, this phenomenon is neither subgroup-specific nor dependent on a previously incriminated Th epitope in the RSV-A G protein . In stark contrast, BBG2Na did not induce any signs of enhanced pulmonary pathology . In conclusion, our data indicate that BBG2Na, formulated in alum, induces safe and protective immune responses against RSV-B challenge in mice . However, the duration of protective immunity will probably be insufficient to prevent RSV-B infection for the duration of the RSV epidemic season.

Biochem J, 2004 Feb 15, 378(Pt 1), 247 - 55
Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5 and -6; Heimlich G et al.; The pro-apoptotic protein Bax plays a key role in the mitochondrial signalling pathway . Upon induction of apoptosis, Bax undergoes a conformational change and translocates to mitochondrial membranes, where it inserts and mediates the release of cytochrome c from the intermembrane space into the cytosol . However, the domains of Bax that are essential for the induction of cytochrome c release are still elusive . Therefore various Bax deletion mutants were generated and expressed in Escherichia coli . The proteins were then purified in order to delineate the function of the transmembrane domain, the BH3 (Bcl-2 homology 3) domain and the putative pore-forming alpha-helices-5 and -6 . These proteins were used to analyse the mechanism of Bax-induced cytochrome c release from mitochondria . None of the Bax proteins caused cytochrome c release merely through physical perturbation of the mitochondrial outer membrane . The alpha-helices-5 and -6 of Bax were shown to mediate the insertion of the protein into mitochondrial membranes and to be essential for the cytochrome c -releasing activity of Bax . In contrast, neither the transmembrane domain nor a functional BH3 domain is required for the Bax-mediated release of cytochrome c from mitochondria.

Arch Microbiol, 2003 Nov, 180(5), 362 - 6 Epub 2003 Sep 03.
The acrAB locus is involved in modulating intracellular acetyl coenzyme A levels in a strain of Escherichia coli CM2555 expressing the chloramphenicol acetyltransferase (cat) gene; Potrykus J et al.; Recently, an Escherichia coli CM2555 strain was described as sensitive to chloramphenicol when expressing the chloramphenicol resistance gene (cat) from a multicopy plasmid . This sensitivity was linked to dysfunction of the acrA gene, which encodes a component of the AcrAB-TolC multidrug efflux pump . Preliminary data indicate that the sensitivity phenotype might be due to a decline in intracellular acetyl coenzyme A concentration accompanying the reaction catalyzed by chloramphenicol acetyltransferase, the cat-encoded resistance protein . Here, we demonstrate that the acrA dysfunction is the factor impairing the intracellular acetyl coenzyme A levels in the cat-expressing CM2555 strain . This effect might be alleviated by the interplay of proteins constituting two homologous efflux systems: AcrAB-TolC and AcrEF-TolC . However, our results show also that this is a genetic background-specific phenomenon, as the decrease in acetyl coenzyme A level is not evident in a cat-bearing DeltaacrAB derivative of the commonly used strain C600.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 1023 - 8
Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo; Hu XB et al.; 3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes . Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity . Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels . Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus . Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000 . Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum . Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia . So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease . All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 1016 - 22
{Functional characterization of recombinant myostatin and its inhibitory role to chicken muscle development}; Yang W et al.; Myostatin is a recently discovered member of transforming growth factor beta (TGFbeta) superfamily and shares similar structure features with other members of TGFbeta superfamily . For a better understanding of molecular mechanism of myostatin function, the production of C-terminal truncated form of recombinant myostatin protein (rMSTN) in E . coli was previously reported . Herein, the functional role of the recombinant myostatin in regulating myogenesis in a chicken embryonic myoblasts (CEMs) system was determined . By using flow cytometric analysis, the myostatin was found to inhibit cell cycle transition from G1 to S phase and result in a cell cycle arrest at G1 . In addition, myostatin blocked the multi-nucleus myotube formation and caused a decreased expression of the muscle cell differentiation markers (myogenin and MHC) in CEMs . In this study, a rabbit polyclonal antibody against myostatin was produced and high affinity and specificity of this anti-myostatin antibody to recombinant and endogenous myostatin were assayed by Western blot analysis . Further studies showed that the antibody could also recognize the tissue endogenous myostatin of human, mouse and rat . A specific 40 kD band was detected in chicken muscle, which suggested that chicken myostatin might have different splicing pattern . Immunofluorescence assay indicated that myostatin predominantly existed in the cytosol in C2C12 cells . Taken together, the results show that myostatin inhibits chicken muscle cells proliferation and differentiation and down-regulates expression of two differentiation marker gene in CEMs . Remarkably, production of functional recombinant myostatin protein and its specific antibody provides important reagents for unraveling molecular mechanisms underlying myostatin action during myogenesis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 993 - 7
Regulation of aroP expression by tyrR gene in Escherichia coli; Wang JG et al.; tyrR gene encodes a global regulatory protein (TyrR), which plays an important role in the transcriptional regulation of eight transcription units (including tyrR gene itself) whose protein products catalyze key steps in aromatic amino acid biosynthesis and/or transport . The aroP gene encodes an integral membrane protein (AroP) that transports aromatic amino acids through the cell membrane . The transcription of aroP was reported to be repressed by TyrR . In this work, aroP(p) (aroP gene carrying its own promoter), aroP (aroP gene without promoter) and tyrR genes were amplified by PCR from genomic DNA of E . coli K12 and introduced into E . coli WT5 . The expression of aroP and tyrR were detected and the activities of AroP and TyrR were determined . The introduction of either aroP(p) or aroP elevated the strain's transport activity by 1.40 or 1.46-fold respectively . Transformant carrying tyrR gene showed an ATPase activity 1.69-fold compared with the control . When the genes were linked in tandem and co-expressed in a plasmid, the relative AroP transport activity of the strain harboring aroP(p) -tyrR (0.95) was significantly lower than that of aroP-tyrR (1.31) . The results indicated that TyrR might be able to reduce the expression of aroP gene by binding with the aroP promoter region in E.coli.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 986 - 92
Expression of C-peptide multiple gene copies in Escherichia coli and stabilities of C-peptide in aqueous solution; Li SX et al.; A gene fragment encoding three copies of proinsulin C-peptide was synthesized and expressed in E . coli and the recombinant proinsulin C-peptide was produced through site-specific cleavage of the resulting gene products . The fusion protein was expressed at high level, about 80 mg/L, as a soluble product in the cytoplasm . Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant, to obtain about 37.5 mg/L of the fusion protein with 70% purity . Enzymatic digestion by trypsin and carboxypeptidase B of the fusion protein efficiently released native C-peptide, the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L . The good agreement of amino acids composition, together with shown similarities of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant C-peptide obtained in this report was the native human C-peptide . The investigation of the chemical stability of recombinant human C-peptide in aqueous solutions by RP-HPLC was also reported . The degradation of the recombinant C-peptide showed a marked dependence on pH and temperature . The degradation reaction of C-peptide occurred immediately in pH 3 or pH 9 buffered solution . The degradation reaction of C-peptide followed first-order kinetics in pH 3 buffered solution at 37 degrees C or 70 degrees C, only 40.3% of C-peptide was remained after 10 h at 70 degrees C . The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degrees C after 10 h and at pH 7.4 and 70 degrees C after 5 h . 99% and 96% of C-peptide was remained at pH 7.4 and 37 degrees C after 10 h with and without 10 g/L BSA respectively.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14263 - 8 Epub 2003 Nov 12.
Intracellular formation of "undisruptable" dimers of inducible nitric oxide synthase; Kolodziejski PJ et al.; Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases . iNOS is active only as a homodimer . Dimerization of iNOS represents a potentially critical target for therapeutic intervention . In this study, we show that intracellular iNOS forms dimers that are "undisruptable" by boiling, denaturants, or reducing agents . Undisruptable (UD) dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro . UD dimers do not form in Escherichia coli-expressed iNOS and could not be assembled in vitro, which suggests that an in vivo cellular process is required for their formation . iNOS UD dimers are not affected by intracellular depletion of H4B . However, the mutation of Cys-115 (critical for zinc binding) greatly affects the formation of UD dimers . This study reveals insight into the mechanisms of in vivo iNOS dimer formation . UD dimers represent a class of iNOS dimers that had not been suspected . This unanticipated finding revises our understanding of the mechanisms of iNOS dimerization and lays the groundwork for future studies aimed at modulating iNOS activity in vivo.

J Biol Chem, 2004 Jan 30, 279(5), 3382 - 8 Epub 2003 Nov 12.
Histone tail-independent chromatin binding activity of recombinant cohesin holocomplex; Kagansky A et al.; Cohesin, an SMC (structural maintenance of chromosomes) protein-containing complex, governs several important aspects of chromatin dynamics, including the essential chromosomal process of sister chromatid cohesion . The exact mechanism by which cohesin achieves the bridging of sister chromatids is not known . To elucidate this mechanism, we reconstituted a recombinant cohesin complex and investigated its binding to DNA fragments corresponding to natural chromosomal sites with high and low cohesin occupancy in vivo . Cohesin displayed uniform but nonspecific binding activity with all DNA fragments tested . Interestingly, DNA fragments with high occupancy by cohesin in vivo showed strong nucleosome positioning in vitro . We therefore utilized a defined model chromatin fragment (purified reconstituted dinucleosome) as a substrate to analyze cohesin interaction with chromatin . The four-subunit cohesin holocomplex showed a distinct chromatin binding activity in vitro, whereas the Smc1p-Smc3p dimer was unable to bind chromatin . Histone tails and ATP are dispensable for cohesin binding to chromatin in this reaction . A model for cohesin association with chromatin is proposed.

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