Escherichia coli

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2029 - 34
Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies; Daugherty PS et al.; Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications . To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) of between 1.7 and 22.5 base substitutions per gene in a high affinity scFv antibody that binds to the cardiac glycoside digoxigenin . The scFv antibody libraries were displayed on Escherichia coli, and mutant populations were analyzed by flow cytometry . At low to moderate mutation frequencies with an average mutation rate of m </= 8, the fraction of clones exhibiting binding to a fluorescently labeled conjugate of digoxigenin decreased exponentially (r(2) = 0.99), but the most highly mutated library (m = 22.5) had significantly more active clones than expected relative to this trend . A library with a low error rate (m = 1.7), one with moderate error rate (m = 3.8), and the one with high error rate (m = 22.5) were screened for high affinity clones under conditions of identical stringency using fluorescence-activated cell sorting . After several rounds of enrichment, each of the three libraries yielded clones with improved affinity for the hapten . The moderate and high error rate libraries gave rise to clones exhibiting the greatest affinity improvement . Taken together, our results indicate that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain-of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site.

Infect Immun, 2000 Mar, 68(3), 1740 - 5
Lipopolysaccharides of Brucella abortus and Brucella melitensis induce nitric oxide synthesis in rat peritoneal macrophages; Lopez-Urrutia L et al.; Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS . The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein . These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucella in humans.

Infect Immun, 2000 Mar, 68(3), 1731 - 4
Lipopolysaccharide entry in the damaged cornea and specific uptake by polymorphonuclear neutrophils; Schultz CL et al.; Bacterial lipopolysaccharide (LPS) is an important agent of induction of ocular pathology following corneal injury or wearing of contaminated contact lenses . The mechanism of LPS uptake through the corneal epithelium is unclear, and the role played by inflammatory cells in this phenomenon has not been previously assessed . Fluorescein isothiocyanate-labeled LPS from Escherichia coli was deposited onto the abraded corneas of New Zealand White rabbits . Epifluorescence microscopy of living excised corneas revealed diffuse LPS staining in the epithelial and stromal layers only in the vicinity of the abrasion . In addition, specific cellular uptake of LPS was suggested by fluorescence staining of cells along the abrasion site . In a second series of experiments, an anti-CD18 polyclonal antibody was used to block infiltration of polymorphonuclear neutrophils (PMN) into the cornea . In these experiments, a diffuse distribution of fluorescent LPS was still observed along the abrasion, but the specific cellular uptake was abolished . The findings indicate that LPS enters the cornea via diffuse penetration at sites of injury and that specific cellular uptake of LPS occurs within the cornea via PMN which have migrated into the damaged tissue.

Infect Immun, 2000 Mar, 68(3), 1626 - 32
Endotoxin-induced lung inflammation is independent of the complement membrane attack complex; Brauer RB et al.; Several products of the activated complement system are known to modulate endothelial cell function in vitro . It has been shown that the membrane attack complex (MAC) (C5b-C9) can enhance tumor necrosis factor alpha (TNF-alpha)-induced expression of P- and E-selectin and intercellular adhesion molecule type 1 in cell cultures of human umbilical vein endothelial cells . In the present study the potential role of this synegism for lung injury during endotoxin-mediated septic shock in vivo was examined using a model of C6-deficient PVG (C-) (RT1(C)) rats and the congenic PVG (C+) (RT1(C)) strain . Following administration of a high (5 mg/kg) or low (0.5 mg/kg) dose of lipopolysaccharide (LPS) (Escherichia coli O55:B5), we determined the expression of cytokines, chemokines, and adhesion molecules as well as the recruitment of leukocytes in the lung . Challenge with intraperitoneal i.p . injections of LPS resulted in a strong induction of TNF-alpha, interleukin-1alpha/beta, cytokine-induced neutrophil chemoattractant, interferon-inducible protein 10, macrophage inflammatory proteins 1alpha and 2, macrophage chemotactic protein 1, and P-selectin . However, there were no significant differences between PVG (C-) and PVG (C+) rats . Immunoperoxidase staining showed a similar increase of lung infiltration by CD11b/c(+) leukocytes in both rat strains . We therefore conclude that the described synergism between TNF-alpha and the MAC of the complement system on the induction of endothelial adhesion molecules is dispensable for inflammatory processes during endotoxin-mediated septic shock in vivo.

Infect Immun, 2000 Mar, 68(3), 1535 - 41
Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid; Ginns CA et al.; The E3 strain of E . coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field . In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence . Virulence was found to be associated with one large plasmid, pVM01 . Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E . coli strain DH5alpha . The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01 . The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure . This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha . Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract . However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha . Thus, these studies have established that infection of chickens with E . coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore . These findings will facilitate identification of the specific genes required for virulence in these pathogens.

Infect Immun, 2000 Mar, 68(3), 1350 - 8
Identification of novel serine/threonine protein phosphatases in Trypanosoma cruzi: a potential role in control of cytokinesis and morphology; Orr GA et al.; We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B . The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B . The proteins encoded by these genes have been tentatively designated TcPP1alpha and TcPP1beta . Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages . Southern blot analysis suggests that each protein phosphatase 1 gene is present as a single copy in the T . cruzi genome . The complete coding region for TcPP1beta was expressed in Escherichia coli by using a vector, pTACTAC, with the trp-lac hybrid promoter . The recombinant protein from the TcPP1beta construct displayed phosphatase activity toward phosphorylase a, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration {IC(50)}, approximately 2 nM) over okadaic acid (IC(50), approximately 100 nM) . Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T . cruzi epimastigotes . Low concentrations of calyculin A (1 to 10 nM) caused growth arrest . Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division . At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology . Okadaic acid at concentrations up to 1 microM did not result in growth arrest or morphological alterations to T . cruzi epimastigotes . Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of (32)P-labeled phosphorylase a by T . cruzi epimastigotes and metacyclic trypomastigote extracts . These inhibitor studies suggest that in T . cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.

Infect Immun, 2000 Mar, 68(3), 1337 - 49
Characterization of in vitro DNA binding sites of the EUO protein of Chlamydia psittaci; Zhang L et al.; The EUO gene of chlamydia is highly expressed early in the developmental cycle, relative to other genes, but continues to be expressed throughout the active growth phases . The precise function of EUO protein is not known, but it binds to DNA in vitro . In this study, we developed a selection and amplification scheme for identifying chlamydial genomic fragments to which EUO preferentially binds in vitro . The scheme involved mixing recombinant EUO with a Chlamydia psittaci genomic library in a pBluescript plasmid vector in vitro, trapping EUO-bound plasmid clones on filters, and amplifying the clones in Escherichia coli . After nine rounds of enrichment, the EUO binding sites of the three most highly enriched clones were identified by DNase I footprint analysis . All three clones had multiple binding sites of various sizes with no clear distinguishing feature other than they were AT-rich and were usually not located in putative promoter regions . We used limited site-specific mutagenesis to characterize the strongest binding site of the most-highly-enriched clone, which represented about 50% of the population after nine rounds . This mutagenesis identified a core binding site of 15 nucleotides (nt) whose sequence was used to find related sequences within each of the strong binding sites in the other two clones . Using the frequency of bases at specific positions within this group of sequences as a guide, we carried out trial-and-error searching with many related sequences, eliminating those which identified nonfootprinted sites . This process led us to the consensus 15-nt sequence AHGAAAWVTYTWDAY, which, when allowing two mismatches, picked out all of the strong binding sites and no nonfootprinting sites within the three enriched clones . This sequence may be useful for predicting additional possible EUO binding sites in the chlamydial genome.

Science, 2000 Feb 18, 287(5456), 1232 - 9
Crystal structure of the ribonucleoprotein core of the signal recognition particle; Batey RT et al.; The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion . We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove . Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo . The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.

Biochemistry, 2000 Feb 22, 39(7), 1870 - 8
Expression and membrane assembly of a transmembrane region from Neu; Jones DH et al.; Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways . The present work addresses problems surrounding expression, isolation, secondary structure recovery, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy . We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes . A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli . The sequence also contained 11-12 amino acids from each side of the transmembrane domain . The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product . This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage . Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification . The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption losses . Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior . Dissolution in certain acidic organic solvents led to material with increased alpha-helix content, which behaved in detergent as mixtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes . For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression . The same acidic organic solvents used to dissolve and dissociate expressed transmembrane peptides proved invaluable for their assembly into lipid bilayers . Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.

Biochemistry, 2000 Feb 22, 39(7), 1784 - 91
Kinetic studies of dimeric Ncd: evidence that Ncd is not processive; Foster KA et al.; Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules . The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein {Chandra, R., Salmon, E . D., Erickson, H . P., Lockhart, A., and Endow, S . A . (1993) J . Biol . Chem . 268, 9005-9013} . Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule . The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N . ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1) . Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site . ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1) . The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's . However, there are two major differences between the mechanisms of Ncd and kinesin . In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover . Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.

Biochemistry, 2000 Feb 22, 39(7), 1725 - 33
Identity of tRNA for yeast tyrosyl-tRNA synthetase: tyrosylation is more sensitive to identity nucleotides than to structural features; Fechter P et al.; The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on the presence of modified residues in tRNA(Tyr), although such residues stabilize its structure . Thus, the major tyrosine identity determinants were searched by the in vitro approach using unmodified transcripts produced by T7 RNA polymerase . On the basis of the tyrosylation efficiency of tRNA variants, the strongest determinants are base pair C1-G72 and discriminator residue A73 (the 5'-phosphoryl group on C1, however, is unimportant for tyrosylation) . The three anticodon bases G34, U35, and A36 contribute also to the tyrosine identity, but to a lesser extent, with G34 having the most pronounced effect . Mutation of the GUA tyrosine anticodon into a CAU methionine anticodon, however, leads to a loss of tyrosylation efficiency similar to that obtained after mutation of the C1-G72 or A73 determinants . Transplantation of the six determinants into four different tRNA frameworks and activity assays on heterologous Escherichia coli and Methanococcus jannaschii tRNA(Tyr) confirmed the completeness of the tyrosine set and the eukaryotic character of the C1-G72 base pair . On the other hand, it was found that tyrosine identity in yeast does not rely on fine architectural features of the tRNA, in particular the size and sequence of the D-loop . Noticeable, yeast TyrRS efficiently charges a variant of E . coli tRNA(Tyr) with a large extra-region provided its G1-C72 base pair is changed to a C1-G72 base pair . Finally, tyrosylation activity is compatible with a +1 shift of the anticodon in the 3'-direction but is strongly inhibited if this shift occurs in the opposite 5'-direction.

Biochemistry, 2000 Feb 22, 39(7), 1655 - 74
Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase; Harris TK et al.; The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus . The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site {Lin, J . , Abeygunawardana, C., Frick, D . N., Bessman, M . J., and Mildvan, A . S . (1997) Biochemistry 36, 1199-1211} . To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined . The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively . Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39 . For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex . The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max) . The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pH-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat) . Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both . A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M(2+) complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn(2+) at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn(2+), respectively, than the wild type enzyme as detected by 1/T(1) values of water protons, consistent with the loss of a metal ligand . However, the K(m) values of Mg(2+) and Mn(2+) indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex . Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M(2+) complexes . Two-dimensional (1)H-(15)N HSQC and three-dimensional (1)H-(15)N NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues . Structural changes in the kinetically more damaged E44D mutant detected in (1)H-(15)N HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region . (1)H-(15)N HSQC titrations of the E53Q, E56Q, and E44D mutants with dGTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site . (ABSTRACT TRUNCATED)

Biochemistry, 2000 Feb 22, 39(7), 1604 - 12
The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle; Klein DJ et al.; The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy . The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses . The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure . The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment . The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain . However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin . A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein {Gao, Y., et al . (1998) Protein Sci . 7, 2265-2280} . However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC . The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.

Hum Antibodies, 1999, 9(3), 165 - 70
From IgG monoclonals to IgM-like molecules; Ernst M et al.; One problem in blood group testing is that IgG monoclonal antibodies, in contrast to IgM, do not usually agglutinate erythrocytes . One of the reasons is the high zeta potential induced by the negative charge of the cell surface . During the last few years, we have produced a series of human monoclonal antibodies by the conventional fusion technique directed against antigens of the Rh blood group system . Some of these monoclonals, especially those directed against Rh-subgroups such as the c-antigen, were mainly of the IgG-subtype and unsuitable for agglutination tests . We have therefore tried to establish a molecular biological method to make IgM-like molecules from IgG monoclonals . From the c-antigen specific human hybridoma BS 240 (IgG subtype), we isolated mRNA that was transcribed into cDNA and then amplified by PCR using family specific primers . The heavy and light chain products were cloned into the pHen vector containing a DNA linker fragment, a myc-tag for identification and a His-tag for purification . After transformation in E.coli and phage rescue with helper phage, the culture supernatant was screened for antigen positive recombinant phage antibodies as a first control for specificity using c-antigen positive erythrocytes and anti-M13 antibodies as bridging antibodies (Coombs technique) . Erythrocytes being negative for the c-antigen served as a negative control . After changing the culture conditions, soluble single chain fragments (scFv) were obtained from the periplasmatic extract . Specificity was shown using the c-antigen positive and negative erythrocytes and the 9E10 antibody (anti-myc) as a bridging antibody . To obtain IgM-like molecules, DNA coding for the specific scFv was cloned into the vector pSTE containing DNA coding for the monomer of core streptavidin . After expression, purification and refolding of the monomer, the core streptavidin combines to form tetrameric structures, termed scFv::strep, that are able to bind biotin as shown using ELISA plates coated with biotinylated BSA . Binding was detected with 9E10 and a peroxidase conjugated secondary antibody . In the agglutination assay, the construct was able to agglutinate c-antigen positive erythrocytes but not the negative erythrocytes . These experiments show that it is possible to construct IgM-like agglutinating molecules from cells containing secreting IgG antibodies . Experiments employing human antibody libraries instead of hybridoma cell lines are now in progress.

Int J Mol Med, 2000 Mar, 5(3), 275 - 8
Regression of experimental liver tumor after distant intra-hepatic injection of cytosine deaminase-expressing tumor cells and 5-fluorocytosine treatment; Pierrefite-Carle V et al.; Cytosine deaminase (CD) gene of E . coli converts the non-toxic compound 5-fluorocytosine (5-FC) into 5-fluorouracil . We have introduced a vector expressing the CD gene in a rat colon carcinoma cell line . Expression of the CD gene confers 5-FC sensitivity to these cells in vitro and in vivo . In a bifocal model consisting in a simultaneous engrafment of a CD+ tumor on one lobe of the liver and a wild-type parental tumor on the opposite lobe, treatment with 5-FC results in regression of both type of tumors, indicating the existence of a distant bystander effect.

RNA, 2000 Feb, 6(2), 220 - 32
Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data; Joseph S et al.; The many interactions of tRNA with the ribosome are fundamental to protein synthesis . During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA . The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome . Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement . We used Fe(II) tethered to the 5' end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5' end of deacylated tRNA(Phe) to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome . We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms . The two tRNAs are constrained to the S configuration with an angle of about 45 degrees between the respective planes of the molecules . The terminal phosphates of 3'CCA are separated by 23 A when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

Nature, 2000 Feb 10, 403(6770), 617 - 22
Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold; Altamirano MM et al.; In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions . The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold . Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold . By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase . We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant . Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.

J Med Invest, 1999 Aug, 46(3-4), 186 - 91
Molecular genetic analysis of pyridoxine-nonresponsive homocystinuric siblings with different blood methionine levels during the neonatal period; Chen S et al.; Two mutations in the cystathionine beta-synthase (CBS) gene were found in two Japanese siblings with pyridoxine non-responsive homocystinuria who had different methionine levels in their blood during the neonatal period . Both patients were compound heterozygotes of two mutant alleles: one had an A-to-G transition at nucleotide 194 (A194 G) that caused a histidine-to-arginine substitution at position 65 of the protein (H65R), while the other had a G-to-A transition at nucleotide 346 (G346A) which resulted in a glycine-to-arginine substitution at position 116 of the protein (G116R) . The two mutant proteins were separately expressed in Escherichia coli, and they completely lacked catalytic activity . Despite their identical genotypes and almost equal protein intake, these siblings showed different levels of blood methionine during the neonatal period, suggesting that the level of methionine in blood is determined not only by the defect in the CBS gene and protein intake, but also by the activity of other enzymes involved in methionine and homocysteine metabolism, especially during the neonatal period . Therefore, high-risk newborns who have siblings with homocystinuria, even if the level of methionine in their blood is normal in a neonatal mass screening, should be followed up and diagnosed by an assay of enzyme activity or a gene analysis so that treatment can be begun as soon as possible to prevent the development of clinical symptoms . In addition, a new, more sensitive method for the mass screening of CBS deficiency in neonates should be developed.

Protein Expr Purif, 2000 Mar, 18(2), 221 - 8
Expression of functional recombinant antibody molecules in insect cell expression systems; Reavy B et al.; Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli . DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines . A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus . However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence . The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium . Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues . Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells . This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain .

Protein Expr Purif, 2000 Mar, 18(2), 175 - 81
Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli; Tong L et al.; HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus . To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium . Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium . The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA) . After IPTG induction, a biologically active rAFP was expressed . The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm . After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L . The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC . Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product . The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function .

Protein Expr Purif, 2000 Mar, 18(2), 121 - 32
Production of fluorescent single-chain antibody fragments in Escherichia coli; Schwalbach G et al.; We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli . The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP) . The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination . The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter . Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen . Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities . The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy . When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments . This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays .

Plasmid, 2000 Mar, 43(2), 159 - 65
Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism; Mendes MV et al.; A cryptic plasmid, pSNA1, has been identified in the pimaricin-producing Streptomyces natalensis strain ATCC 27448 . pSNA1 has been mapped with restriction endonucleases and its complete nucleotide sequence was determined . The circular DNA molecule is 9367 bp in length and has a 71.3% G+C content . Its estimated copy number is 30 . Analysis of the sequence and codon preferences indicated that pSNA1 contains seven open reading frames {encoding peptides larger than 90 amino acid (aa) residues}, ORF 1 to ORF 7, located on both strands of pSNA1 . ORF 3 codes for a protein (476 aa) that shows high sequence similarity to replication-associated proteins in Streptomyces plasmids known to replicate via the rolling circle mechanism . Accumulation of single-strand intermediates further indicates that pSNA1 replicates via the rolling circle replication model . ORF 1 encodes a polypeptide of 246 aa that shares homology with KorA proteins encoded by other streptomycete plasmids . ORF 4 (SpdA) codes for a protein (161 aa) possibly involved in intramycelial plasmid transfer . Protein encoded by ORF 2 (309 aa) shares homology with a Streptomyces protein (SpdB2) also involved in plasmid spreading .

Plasmid, 2000 Mar, 43(2), 149 - 52
The pilL and pilN genes of IncI1 plasmids R64 and ColIb-P9 encode outer membrane lipoproteins responsible for thin pilus biogenesis; Sakai D et al.; The predicted amino acid sequences of the pilL and pilN genes, required for the thin pilus formation of IncI1 plasmids R64 and ColIb-P9, contain N-terminal lipoprotein signal peptide motifs . The pilL and pilN products were labeled with {(3)H}palmitic acid as 38- and 57-kDa proteins, respectively, indicating that they are lipoproteins . Both PilL and PilN were localized to the outer membrane .

J Mol Biol, 2000 Mar 3, 296(4), 1053 - 63
Thermodynamics of DNA binding and condensation: isothermal titration calorimetry and electrostatic mechanism; Matulis D et al.; The thermodynamics of binding of the trivalent cations cobalt hexammine and spermidine to plasmid DNA was studied by isothermal titration calorimetry . Two stages were observed in the course of titration, the first attributed to cation binding and the second to DNA condensation . A standard calorimetric data analysis was extended by applying an electrostatic binding model, which accounted for most of the observed data . Both the binding and condensation reactions were entropically driven (TDeltaS approximately +10 kcal/mol cation) and enthalpically opposed (DeltaH approximately +1 kcal/mol cation) . As predicted from their relative sizes, the binding constants of the cations were indistinguishable, but cobalt hexammine had a much greater DNA condensing capacity because it is more compact than spermidine . The dependence of both the free energy of cobalt hexammine binding and the critical cobalt hexammine concentration for DNA condensation on temperature and monovalent cation concentration followed the electrostatic model quite precisely . The heat capacity changes of both stages were positive, perhaps reflecting both the temperature dependence of the dielectric constant of water and the burial of polar surfaces . DNA condensation occurred when about 67 % of the DNA phosphate charge was neutralized by cobalt hexammine and 87 % by spermidine . During condensation, the remaining DNA charge was neutralized .

J Mol Biol, 2000 Mar 3, 296(4), 1001 - 15
Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation; Aleshin AE et al.; Hexokinase I, the pacemaker of glycolysis in brain tissue, is composed of two structurally similar halves connected by an alpha-helix . The enzyme dimerizes at elevated protein concentrations in solution and in crystal structures; however, almost all published data reflect the properties of a hexokinase I monomer in solution . Crystal structures of mutant forms of recombinant human hexokinase I, presented here, reveal the enzyme monomer for the first time . The mutant hexokinases bind both glucose 6-phosphate and glucose with high affinity to their N and C-terminal halves, and ADP, also with high affinity, to a site near the N terminus of the polypeptide chain . Exposure of the monomer crystals to ADP in the complete absence of glucose 6-phosphate reveals a second binding site for adenine nucleotides at the putative active site (C-half), with conformational changes extending 15 A to the contact interface between the N and C-halves . The structures reveal distinct conformational states for the C-half and a rigid-body rotation of the N-half, as possible elements of a structure-based mechanism for allosteric regulation of catalysis .

J Mol Biol, 2000 Mar 3, 296(4), 951 - 9
Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase; Borgstahl GE et al.; Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide . We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD) . Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K . Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand . The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions . The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand .

J Med Virol, 2000 Apr, 60(4), 379 - 86
Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein; Li F et al.; A recombinant subunit antigen (ORF2.1), representing the carboxy-terminal 267 amino acids of the 660-amino-acid hepatitis E virus (HEV) capsid protein, was expressed in Escherichia coli and used for the immunisation of rats . Purified antigen formulated with either Aluminium Hydroxide Gel Adjuvant (Alum) or Titermax gave high and equivalent levels of antibody after three doses . Responses to two doses of 15, 75, or 150 microg antigen, formulated with Alum and given at 0 and 4 weeks, were also equivalent by 17 weeks after immunisation . Rats initially developed antibody to a wide range of linear epitopes in the ORF2.1 region, but by 27 weeks the predominant response detected by Western immunoblotting was restricted to the conformational epitope unique to ORF2.1 {Li et al . (1997) Journal of Medical Virology 52:289-300}, a pattern that was also observed when comparing acute-phase patient serum samples with serum samples from convalescing patients . Antibody from immunised rats blocked the majority of patients' serum reactivity in enzyme-linked immunosorbent assay against both ORF2.1 (57-92% inhibition) and virus-like particles of HEV produced using the baculovirus system (74-97% inhibition) . Together, these results suggest that the ORF2.1 subunit vaccine induces an antibody response against immunodominant, conformational epitopes in the viral capsid, which largely mimics that seen in convalescent patients, who are presumed to be immune to HEV infection .

Transfusion, 2000 Feb, 40(2), 245 - 51
Prevalence of the newly described human circovirus, TTV, in United States blood donors; Handa A et al.; BACKGROUND: A novel nonenveloped single-stranded circular DNA virus (TTV) was recently identified . The prevalence of TTV in blood donors in the United States is, however, still unclear . STUDY DESIGN AND METHODS: Viral DNA was detected in US blood donors from five cities by using two sets of TTV primers: NG059/NG061/NG063 primers, which amplified the conserved region of strains 1 and 2, and T801/T935 primers, which amplified the 5' end region of the TTV sequence . A TTV antibody assay system was based on the detection of the truncated open reading frame (ORF)-1 (amino acids 1-411) from type 1b . The truncated ORF-1 was expressed as a fusion protein in Escherichia coli, and the fusion protein was used as the antigen in the antibody assay system . RESULTS: Viremia was detected in 21 (8 . 4%) of 250 donors by use of NG059/NG061/NG063 primers and 104 (41 . 6%) of 250 by use of T801/T935 primers . There was little correlation among the assays, which suggests the preferential detection of different strains with the different primers . TTV antibody was detected in 38 of 100 donors: 32 (84%) of 38 with concurrent TTV viremia and 6 (16%) of 38 without TTV viremia . TTV viremia and/or TTV antibody-positive samples were detected in 52 (52%) of 100 of US blood donors . CONCLUSION: Evidence of infection or exposure to TTV appears to be common among blood donors in United States.

Poult Sci, 2000 Jan, 79(1), 26 - 32
Effects of moniliformin on performance and immune function of broiler chicks; Li YC et al.; Three trials were conducted to evaluate the effect of moniliformin (M) on performance and immune function in chicks . Day-old chicks were randomly assigned to four dietary treatments (0, 50, 75, or 100 mg M/kg diet) . In Trial 1, chicks were placed on treatments for 3 wk and were injected intravenously with 4.6 x 10(6) Escherichia coli on Day 21 . Blood samples were collected at 60, 120, and 180 min after inoculation, and liver, spleen, and lung were collected at 180 min postinjection . Compared with control chicks, chicks fed 75 and 100 mg M/ kg diet had higher (P < 0.05) numbers of E . coli colonies in the circulation, liver, and spleen . In Trial 2, chicks were placed on diets for 4 wk and were injected with 0.5 mL Newcastle disease virus (NDV) vaccine intramuscularly on Weeks 2 and 3 of the experiment . The primary and secondary anti-NDV antibody titers were measured 7 d after each injection . Chicks fed 100 mg M/kg diet had lower (P < 0.05) secondary antibody titers than did control chicks . In Trial 3, lymphocyte proliferation in chicks exposed to M in vivo and in vitro was determined . Results of the in vivo study showed that cell proliferation in response to mitogens from control- and M-fed chicks did not differ (P > 0.05) . For the in vitro study, lymphocyte proliferation decreased linearly (P < 0.01) with increased concentrations of M . In all three trials, chicks fed 100 mg M/kg diet had lower (P < 0.05) feed intake and weight gain than did control chicks . Data from the current study suggested that M decreased performance and immune response in chicks at the level of 75 mg/kg diet.

Am J Vet Res, 2000 Feb, 61(2), 125 - 8
Transformation and transposition of the genome of Mycobacterium marinum; Talaat AM et al.; OBJECTIVE: To develop and evaluate protocols for genetic manipulations (transformation and transposition) of the fish pathogen, Mycobacterium marinum . SAMPLE POPULATION: Isolates of M . marinum obtained from fish and humans . PROCEDURE: Electrocompetent cells were prepared from isolates of M . marinum grown to various growth phases at several temperatures and with or without the addition of ethionamide or cycloheximide . Mycobacterial cells were transformed by electroporation with a replicative Escherichia coli-mycobacteria shuttle vector (pYUB18) as well as suicide vectors (pYUB285 and pUS252) that carried transposable elements (IS1096 and IS6110, respectively) . Mutants from both isolates of M . marinum were recovered on 7H10 agar plates supplemented with kanamycin . Transformation and transposition efficiencies for various protocols were compared . Southern hybridization analysis was performed on mycobacterial mutants to confirm transposition events . RESULTS: Competent cells prepared at room temperature (23-25 C) from organisms in late-exponential growth phase yielded higher transposition efficiency, compared with cells prepared at 4 C or from organisms in early- or mid-exponential growth phase . Naturally developing kanamycin-resistant colonies of M . marinum were not detected . Only the IS1096-derived transposition was able to efficiently mutate M . marinum . Southern hybridization of M . marinum mutants revealed random integration of IS 1096 into the M . marinum genome . CONCLUSIONS: Transposition and transformation efficiencies were comparable, suggesting that the limiting factor in transposition is the transformation step . Most of the experiments resulted in transposition of IS1096; however, better approaches are needed to improve transposition efficiency.

J Biomol NMR, 1999 Dec, 15(4), 335 - 8
Line narrowing in spectra of proteins dissolved in a dilute liquid crystalline phase by band-selective adiabatic decoupling: application to 1HN-15N residual dipolar coupling measurements; Vander Kooi CW et al.; Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination . However, partial orientation of protein samples also causes severe 1H line broadening resulting from residual 1H-1H dipolar couplings . In this communication we show that band-selective 1H homonuclear decoupling during data acquisition is an efficient way to suppress residual 1H-1H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment . As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond 1HN-15N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles.

Biochim Biophys Acta, 2000 Feb 29, 1490(3), 245 - 58
Expression and characterization of the human mitochondrial leucyl-tRNA synthetase; Bullard JM et al.; A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases . Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids . Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins . The mature form of this protein has been cloned and expressed in Escherichia coli . Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa . The human mtLeuRS is capable of aminoacylating E . coli tRNA(Leu) . Its activity is inhibited at high levels of either monovalent or divalent cations . K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.

J Neurosci, 2000 Mar 1, 20(5), 1694 - 700
Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-alpha knock-out mice; Singh M et al.; We have shown previously in the developing cerebral cortex that estrogen elicits the rapid and sustained activation of multiple signaling proteins within the mitogen-activated protein (MAP) kinase cascade, including B-Raf and extracellular signal-regulated kinase (ERK) . Using estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, we addressed the role of ER-alpha in mediating this action of estrogen in the brain . 17beta-Estradiol increased B-Raf activity and MEK (MAP kinase/ERK kinase)-dependent ERK phosphorylation in cerebral cortical explants derived from both ERKO and their wild-type littermates . The ERK response was stronger in ERKO-derived cultures but, unlike that of wild-type cultures, was not blocked by the estrogen receptor antagonist ICI 182,780 . Surprisingly, both the ER-alpha selective ligand 16alpha-iodo-17beta-estradiol and the ER-beta selective ligand genistein failed to elicit ERK phosphorylation, suggesting that a different mechanism or receptor may mediate estrogen-induced ERK phosphorylation in the cerebral cortex . Interestingly, the transcriptionally inactive stereoisomer 17alpha-estradiol did elicit a strong induction of ERK phosphorylation, which, together with the inability of the ER-alpha- and ER-beta-selective ligands to elicit ERK phosphorylation, and of ICI 182,780 to block the actions of estradiol in ERKO cultures, supports the hypothesis that a novel, estradiol-sensitive and ICI-insensitive estrogen receptor may mediate 17beta-estradiol-induced activation of ERK in the brain.

Biol Reprod, 2000 Mar, 62(3), 606 - 15
Expression of human proacrosin in Escherichia coli and binding to zona pellucida; Furlong LI et al.; Proacrosin is a multifunctional protein present in the sperm acrosome . This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity . The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors . In the pGEX system, expression of the full-length fusion protein was not detected . In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42-44 kDa) was recognized by a monoclonal antibody to human acrosin, AcrC5F10 . A 32-34-kDa protein (Rec-30), not recognized by AcrC5F10 on Western blots, was the major expression product . Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression products as were Rec-40 and Rec-30, and truncated products from the C terminus were detected in the soluble fraction . Rec-40 and Rec-30 coexisted at any culture time tested . Immune serum raised against Rec-30 (AntiRec-30) stained the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts . Amino acid sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-terminal fragments of proacrosin . The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous (125)I-zona pellucida glycoproteins.

Biochemistry, 2000 Feb 29, 39(8), 2106 - 22
Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein . stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues; Bujalowski W et al.; The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique . The experiments have been performed with fluorescent ATP and ADP analogues bearing the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, epsilonAMP-PNP and epsilonADP . Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four-steps . The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps . This mechanism is described by the following equation: {equation in text} . The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system . Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide . It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)(1) . Moreover, the value of the bimolecular rate constant, k(1), is 3-4 orders of magnitude lower than the value expected for the diffusion-controlled reaction . These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex . The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates . The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding . The significance of these results for the functioning of the DnaB helicase is discussed.

Biochemistry, 2000 Feb 29, 39(8), 1924 - 34
Crystallographic studies of the interactions of Escherichia coli lytic transglycosylase Slt35 with peptidoglycan; van Asselt EJ et al.; Lytic transglycosylases catalyze the cleavage of the beta-1, 4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydro bond in the MurNAc residue . To understand the reaction mechanism of Escherichia coli lytic transglycosylase Slt35, three crystal structures have been determined of Slt35 in complex with two different peptidoglycan fragments and with the lytic transglycosylase inhibitor bulgecin A . The complexes define four sugar-binding subsites (-2, -1, +1, and +2) and two peptide-binding sites in a large cleft close to Glu162 . The Glu162 side chain is between the -1 and +1 sugar-binding sites, in agreement with a function as catalytic acid/base . The complexes suggest additional contributions to catalysis from Ser216 and Asn339, residues which are conserved among the MltB/Slt35 lytic transglycosylases.

Biochemistry, 2000 Feb 15, 39(6), 1522 - 31
Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme; Fleming SM et al.; 2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family . The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I . M . J., and Bugg, T . D . H . (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation . Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes . In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate . Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl . These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine . The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.

Biochemistry, 2000 Feb 15, 39(6), 1499 - 514
Structural consequences of b- to c-type heme conversion in oxidized Escherichia coli cytochrome b562; Arnesano F et al.; An NMR characterization of the 98Arg --> Cys variant of iron (III)-containing cytochrome b562 from Escherichia coli has been performed and the solution structure obtained . This variant has a covalent bond between the heme and Cys 98, thus mimicking the heme binding in cytochrome c . The R98C cytochrome is shown to have a significantly increased stability, compared to that of wild type, toward thermal and chemical denaturation . In water at 20 degrees C it is 5.60 kJ mol-1 more stable than the WT protein, measured by equilibrium guanidine hydrochloride denaturation . The structure has been obtained through two-dimensional total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments and through three-dimensional NOESY-15N heteronuclear multiple quantum coherence (HMQC) . By these methods, 85% of protons and 100% of backbone nitrogens were assigned . 2145 meaningful nuclear Overhauser effects (NOEs) (20 NOEs per residue), 45 backbone 3J values, and 397 pseudocontact shifts were used to obtain a family of 35 members, which were then energy-minimized . The root-mean-square deviation (RMSD) with respect to the average structure is 0.50 +/- 0.07 for the backbone and 1.01 +/- 0.08 for the heavy atoms . The magnetic anisotropy resulting from analysis of the pseudocontact shifts indicates an anisotropy that is an intermediate between that of the wild-type, which is the smallest, and cytochrome c . The g values confirm a higher anisotropy of the variant with respect to the wild-type protein . The chirality of the heme 2 alpha carbon is the same as that in all naturally occurring cytochromes c . The overall secondary structure and tertiary structure are very similar to the wild type . The removal of Arg 98 causes a change in the pH-dependent properties . The pKa, proposed to be due to deprotonation of the coordinated histidine, is 1.5 units higher than in the wild type, consistent with the lack of the positive charge of Arg 98 close to the ionizable group . This is further support for the coordinated histidine being the titratable group with an alkaline pKa in the wild-type protein . The pattern of the shifts of the heme methyl groups is different than in the wild-type protein, presumably due to alteration of the electronic structure by the presence of the covalent bond between the protein and the heme . The difference in stability between the variant and wild-type protein is discussed in terms of the structural information.

Biochemistry, 2000 Feb 15, 39(6), 1455 - 61
Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence; Fischer CJ et al.; The room-temperature tryptophan (Trp) phosphorescence lifetime is sensitive to details of the local environment and has been shown to increase significantly in some proteins following H-D exchange . Careful analysis of the phosphorescence lifetime distribution of Trp 109 in Escherichia coli alkaline phosphatase (AP) in solution as a function of time during the H-D exchange shows that this process corresponds to a two-state reaction resulting from the deuteration of a single, specific hydrogen in the core of the protein . The absence of a pH dependence of the exchange rate suggests that the exchange is not an EX2 process, and therefore, a certain degree of unfolding is required for exchange to occur . This discovery opens up the use of phosphorescence-detected hydrogen exchange as a sensitive tool for monitoring the local susceptibility and activation energy for exchange in proteins having a phosphorescent Trp and, for example, for studying the effects of local mutations upon that susceptibility.

Biochemistry, 2000 Feb 15, 39(6), 1420 - 6
Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function; Shroff NP et al.; An autosomal dominant congenital cataract in human is associated with mutation of Arg-116 to Cys (R116C) in alpha A-crystallin . To investigate the molecular basis of cataract formation, rat alpha A-crystallin cDNA was cloned into pET-23d(+), and the site-directed mutants S142C (similar to wild-type human alpha A) and R116C/S142C or R116C (similar to human R116C variant) were generated . These were expressed in E . coli and the recombinant alpha A-crystallins purified by Sephacryl size-exclusion chromatography . The chaperone-like function of mutant R116C determined at 37 degrees C with insulin and alcohol dehydrogenase as target proteins was about 40% lower than those of wild-type and mutant S142C . Based on size-exclusion chromatography data, the oligomeric size of the R116C mutant was about 2000 kDa at 25 degrees C, 1400 kDa at 37 degrees C, and 900 kDa at 45 degrees C . In comparison, alpha A-wild-type and alpha A-S142C ranged from 477 to 581 kDa . Heat stability studies corroborated the effect of temperature on the dynamic quaternary structure of the R116C mutant . Circular dichroism spectra showed secondary and tertiary structural changes, and ANS fluorescence spectra showed loss of surface hydrophobicity in the R116C mutant . These findings suggest that the molecular basis for the congenital cataract with the alpha A-R116C mutation is due to the generation of a highly oligomerized alpha A-crystallin having a modified structure and decreased chaperone-like function.

Biochemistry, 2000 Feb 15, 39(6), 1338 - 45
Active-site-directed photolabeling of the melibiose permease of Escherichia coli; Ambroise Y et al.; Covalent photolabeling of the melibiose permease (MelB) of Escherichia coli has been undertaken with the sugar analogue {(3)H}-p-azidophenyl alpha-D-galactopyranoside ({(3)H}-alpha-PAPG) with the purpose of identifying the domains forming the MelB sugar-binding site . We show that alpha-PAPG is a high-affinity substrate of MelB (K(d) = 1 x 10(-)(6) M) . Its binding to or transport by MelB is Na-dependent and is competitively prevented by melibiose or by the high-affinity ligand p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) . Membrane vesicles containing overexpressed histidine-tagged recombinant MelB were photolabeled in the presence of {(3)H}-alpha-PAPG by irradiation with UV light (lambda = 250 nm) . Eighty-five percent of the radioactivity covalently associated with the vesicles was incorporated in a polypeptide corresponding to MelB monomer . MelB labeling was completely prevented by an excess of melibiose or alpha-NPG during the assay . Radioactivity analysis of CNBr cleavage or limited proteolysis products of the purified {(3)H}-alpha-PAPG-labeled transporter suggests that several domains of MelB are targets for labeling . One of the labeled CNBr cleavage products is a peptide with an apparent molecular mass of 5.5 kDa . It is shown that (i) its amino acid sequence is that of the Asp124-Met181 domain of MelB (7.5 kDa), which includes the cytoplasmic loop 4-5 connecting helices IV and V, the hydrophobic helix V, and the outer loop connecting helices V-VI, and (ii) that Arg141 in loop 4-5 is the only labeled amino acid of this peptide . Labeling of loop 4-5 provides independent evidence that this specific domain plays a significant role in MelB transport . Comparison with the well-characterized equivalent domain of LacY suggests that sugar transporters with similar structure and substrate specificity may have conserved domains involved in sugar recognition.

Biochemistry, 2000 Feb 15, 39(6), 1285 - 93
Mechanistic analysis of the argE-encoded N-acetylornithine deacetylase; Javid-Majd F et al.; The E . coli argE-encoded N-acetyl-L-ornithine deacetylase has been cloned, expressed, and purified in high yield . The substrate specificity of the enzyme is relatively broad, with a number of alpha-N-acyl-L-amino acids exhibiting activity, including both alpha-N-acetyl- and alpha-N-formylmethionine that exhibit higher activity than alpha-N-acetyl-L-ornithine . Sequence homolgy suggests that the enzyme is a member of the metal-dependent aminoacylase family, and the purified enzyme contains a single atom of zinc per monomer . The activity of this enzyme can be increased greater than 2-fold by the addition of zinc, or 8-fold by the addition of cobalt . This suggests that the enzyme can accommodate two metal ions at the active site . The pH dependence of the kinetic parameters has been determined and revealed the presence of two enzymic groups, one functioning as a general base and one functioning as a general acid . Solvent kinetic isotope effects on the hydrolysis of N-acetylornithine have been determined, and a linear proton inventory suggests that a single proton transfer occurs in a partially rate-limiting step . A chemical mechanism is proposed and compared with other mechanisms determined for other members of the aminoacylase family.

Biochemistry, 2000 Feb 15, 39(6), 1263 - 73
Structure of a NifS homologue: X-ray structure analysis of CsdB, an Escherichia coli counterpart of mammalian selenocysteine lyase; Fujii T et al.; Escherichia coli CsdB, a NifS homologue with a high specificity for L-selenocysteine, is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that belongs to aminotransferases class V in fold-type I of PLP enzymes and catalyzes the decomposition of L-selenocysteine into selenium and L-alanine . The crystal structure of the enzyme has been determined by the X-ray crystallographic method of multiple isomorphous replacement and refined to an R-factor of 18.7% at 2.8 A resolution . The subunit structure consists of three parts: a large domain of an alpha/beta-fold containing a seven-stranded beta-sheet flanked by seven helices, a small domain containing a four-stranded antiparallel beta-sheet flanked by three alpha-helices, and an N-terminal segment containing two alpha-helices . The overall fold of the subunit is similar to those of the enzymes belonging to the fold-type I family represented by aspartate aminotransferase . However, CsdB has several structural features that are not observed in other families of the enzymes . A remarkable feature is that an alpha-helix in the lobe extending from the small domain to the large domain in one subunit of the dimer interacts with a beta-hairpin loop protruding from the large domain of the other subunit . The extended lobe and the protruded beta-hairpin loop form one side of a limb of each active site in the enzyme . The most striking structural feature of CsdB lies in the location of a putative catalytic residue; the side chain of Cys364 on the extended lobe of one subunit is close enough to interact with the gamma-atom of a modeled substrate in the active site of the subunit . Moreover, His55 from the other subunit is positioned so that it interacts with the gamma- or beta-atom of the substrate and may be involved in the catalytic reaction . This is the first report on three-dimensional structures of NifS homologues.

Biochemistry, 2000 Feb 15, 39(6), 1189 - 98
Kinetic studies of the mechanism of carbon-hydrogen bond breakage by the heterotetrameric sarcosine oxidase of Arthrobacter sp . 1-IN; Harris RJ et al.; The reaction of heterotetrameric sarcosine oxidase (TSOX) of Arthrobactor sp . 1-IN has been studied by stopped-flow spectroscopy, with particular emphasis on the reduction of the enzyme by sarcosine . Expression of the cloned gene encoding TSOX in Escherichia coli enables the production of TSOX on a scale suitable for stopped-flow studies . Treatment of the enzyme with sulfite provides the means for selective formation of a flavin-sulfite adduct with the covalent 8alpha-(N(3)-histidyl)-FMN . Formation of the sulfite-flavin adduct suppresses internal electron transfer between the noncovalent FAD (site of sarcosine oxidation) and the covalent FMN (site of enzyme oxidation) and thus enables detailed characterization of the kinetics of FAD reduction by sarcosine using stopped-flow methods . The rate of FAD reduction displays a simple hyperbolic dependence on sarcosine concentration . Studies in the pH range 6.5-10 indicate there are no kinetically influential ionizations in the enzyme-substrate complex . A plot of the limiting rate of flavin reduction/the enzyme-substrate dissociation constant (k(lim)/K(d)) versus pH is bell-shaped and characterized by two macroscopic pK(a) values of 7.4 +/- 0.1 and 10.4 +/- 0.2: potential candidates for the two ionizable groups are discussed with reference to the structure of monomeric sarcosine oxidase (MSOX) . The kinetic data are discussed with reference to potential mechanisms for the oxidation of amine molecules by flavoenzymes . Additionally, kinetic isotope effect studies of the rate of C-H bond breakage suggest that a ground-state quantum tunneling mechanism for H-transfer, facilitated by the low-frequency thermal motions of the protein molecule, accounts for C-H bond cleavage by TSOX . TSOX thus provides another example of C-H bond breakage by ground-state quantum tunneling, driven by protein dynamics {vibrationally enhanced ground-state quantum tunneling (VEGST)}, for the oxidation of amines by enzymes.

Nucleic Acids Res . 2000 Mar 15;28(6):E16.
Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique; Jaruga P et al.; GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases . To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples . Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver . In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations . The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively . It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.

Nucleic Acids Res, 2000 Mar 15, 28(6), 1428 - 38
Functional alpha-fragment of beta-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus; Lin J et al.; PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease . This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript . Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression . To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast . The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli . The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA . This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene . Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.

Nucleic Acids Res, 2000 Mar 15, 28(6), 1374 - 80
Modified constructs of the tRNA TPsiC domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases; Sengupta R et al.; The TPsiC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m(5)C(49), T(54), Psi(55)and m(1)A(58) . U(54)is methylated to m(5)U (T) by m(5)U(54)methyltransferase (RUMT); A(58)is methylated to m(1)A by m(1)A(58)tRNA methyltransferase (RAMT) . RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule . We report that RAMT can recognize and methylate a TSL heptadecamer . To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities . TSLs were synthesized with natural modifications (T(54)and Psi(55)), naturally occurring modifications at unnatural positions (m(5)C(60)), altered sugar puckers (dU(54)and/or dU(55)) or with disrupted U-turn interactions (m(1)Psi(55)or m(1)m(3)Psi(55)) . The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT . The presence of T(54)increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate . Local conformation around U(54)was found to be an important determinant for the activities of both RAMT and RUMT.

J Mol Evol, 2000 Feb, 50(2), 184 - 93
Codon usage in plastid genes is correlated with context, position within the gene, and amino acid content; Morton BR et al.; Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs . This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency . In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E . coli and which may influence translation efficiency . These features are (1) a relatively low codon adaptation at the 5' end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content . All three features are found in plastid genes . First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10-20 codons . Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3' neighboring base is a G . At these sites highly expressed genes are biased toward NNT instead of NNC . Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular . The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property . It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency.

Shi Yan Sheng Wu Xue Bao, 1997 Mar, 30(1), 65 - 71
{Gene expression and distribution in mouse abdominal cavity mediated by adenovirus}; Wu HQ et al.; Infection and expression of recombinant human adenovirus in mouse abdominal cavity was reported . After adenovirus vector Ad/RSV-beta-gal harboring the E . coli lacZ marker gene was injected into mice abdominal cavity, the peritoneal surface of jejunum, ileum, colon, uterus, liver, spleen, stomach, bladder, abdominal wall, diaphragm and testis was found large patches of lacZ-positive cells . But the adenovirus vector was not able to penetrate the peritoneum, as demonstrated by histochemical staining . Another adenovirus vector Ad/RSV-tk harboring the HSV-tk gene was injected into mouse abdominal cavity and the mouse was treated with ACV . No acute toxic reaction was observed . Based on these data, the feasibility of gene therapy of malignant tumor within abdominal cavity with adenovirus mediated TK/GCV system was discussed.

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Mar, 29(1), 11 - 5
{Study on the beta-lactamases of the Escherichia coli HX88108 resistant to ceforperazon}; Zhou L et al.; E . coli HX88108 was isolated from a patient and found to produce plasmid-encode beta-lactamases with conferring highly resistance to ceforperazone(CPZ) . The beta-lactamases of the E . coli HX88108 and transformants pFC, pFT1, pFT2 and pFT3 were studied . The beta-lactamases stability test among 11 beta-lactam antibiotics showed that beta-lactamases from E . coli HX88108 . pFC, pFT1, readily hydrolyzed penicillins, the first, second-generation cephalosporins and CPZ . beta-lactamases of pFT2 and pFT3 hydrolyzed penicillins more strongly than cephalosporins . On the other hand, experiments of inhibiting enzyme were carried out . The results indicated that beta-lactamases of HX88108, pTF1, pFT2 and pFT3 were inhibited by clavulanic acid(CA) and sulbactam (SBT) . Enzyme of pFC was inhibited poorly by CA and SBT . Through isoelectric focusing technique, the PIs were as follows: HX88108 contained three beta-lactamases, of which the PIs were 5.25, 5.3 and 5.6 respectively; the PIs of beta-lactamases from pFT2, pFT3, were 5.3 and 5.6 . pFC and pFT1 were different plasmids encoded beta-lactamases with the same PI 5.25 . The results indicate that the beta-lactamases of E . coli HX88108 may be a new member in TEM farmily.

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Mar, 29(1), 1 - 6
{Cloning and expression of leptospiral protective antigen gene OmpL1 in BCG}; Wan B et al.; This study was intended to produce a new living vaccine against leptospirosis using BCG as vector . Leptospiral outer envelop antigen gene OmpL1 was amplified from the genome of pathogenic leptopira serova Lai 017 by PCR, and cloned in E . coli-BCG shuttle plasmid pY6002 . Recombinant plasmids were isolated by dot blotting with Digoxigeninlabeled OmpL1 gene . After transforming the recombinant plasmids in BCG (Shanghai strain) by electroporation, the genomic DNA of all 21 transformants were prepared and hybridized with OmpL1 . It showed that 6 of the 21 transformants were recombinants in which the OmpL1 gene had been integrated into the genome of BCG . By immunoblotting with OmpL1 infected rabbit antiserum, which was preabsorbed to remove antibody against E . coli and SPA-HRP, three recombinants, pLI1, pLI2 and pLI3, were detected to express OmpL1 protein . The ability of expression is in the order of pLI2 > pLI1 >> plI3 . These studies provide the possibility of further research on the development of highly efficient recombinant vaccines against leptospirosis.

IUBMB Life, 1999 Dec, 48(6), 613 - 8
Single-stranded oligodeoxyribonucleotides are substrates of Fpg protein from Escherichia coli; Ishchenko AA et al.; The interaction of Escherichia coli Fpg protein, which catalyzes excision of several damaged purine bases including 8-oxoguanine (oxoG) from DNA with a set of single- (ss) and double-stranded (ds) 23-mer oligodeoxyribonucleotides (ODNs) containing 8-oxoguanine(s) at various positions, has been investigated . The affinities of different ss ODNs (KM = 0.55-1.3 microM) were shown to be 12-170 times less than those for corresponding ds ODNs (KM = 6-60 nM) . Depending on the position of the oxoG within the ODNs, relative initial rates of conversion of ss substrates may be less than, comparable, or greater than those for ds ODNs . The enzyme can remove 5'-terminal oxoG from ODNs only if the 5'-end is phosphorylated . Fpg does not release oxoG residues from the ultimate and penultimate 3'-terminal positions . Duplexes containing two adjacent oxoG are poor substrates for the glycosylase.

Biotechniques, 2000 Feb, 28(2), 338 - 44
Cell-free synthesis and affinity isolation of proteins on a nanomole scale; Alimov AP et al.; The performance of conventional cell-free gene expression systems based on the Escherichia coli S30 extract can be significantly improved by using expression vectors that encode viral structural elements known to enhance translation in vivo and to protect mRNA from ribonuclease action . The expression vectors reported here are designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus . They can be used in translation, transcription-translation or replication-translation reactions . Depending on its type, the reaction yields up to 40 micrograms per mL, or about 1 nmol of a standard protein . The presence of Strep-tag allows the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions and the entire procedure to be completed within one working day . The results show that standard low-cost, cell-free systems can serve for rapid preparation of purified proteins in amounts that can satisfy a number of needs of a research laboratory.

FEMS Immunol Med Microbiol, 2000 Mar, 27(3), 201 - 10
Prevention of endotoxin-induced lethality in mice by calmodulin kinase activator; Asai Y et al.; Porphyromonas gingivalis strain 381 lipid A showed lower activity in inducing interleukin (IL)-1alpha and IL-1beta production and cytokine mRNA expression than synthetic Escherichia coli lipid A (compound 506) in alveolar macrophages of C57BL/6 mice . Both the lipid As induced tumor necrosis factor alpha in alveolar macrophages and IL-6 in peritoneal macrophages . A calmodulin (CaM) antagonist, W-7, inhibited IL-1beta production and its mRNA expression induced by P . gingivalis lipid A but not compound 506 in alveolar macrophages . A CaM kinase activator reduced the induction of IL-1beta in the serum of mice when administered with compound 506, and protected the mice against the lethal toxicity . The modulation of a variety of intracellular enzymes including the CaM kinase may result in clinical control of endotoxic sepsis.

FEBS Lett, 2000 Feb 18, 468(1), 11 - 4
Amino acids and peptides . LVII . Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase; Joshi S et al.; The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined . The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet) . These data were not consistent with previous reports that both the native peptide isolated from S . solfataricus {Fusi et al . (1993) Eur . J . Biochem . 211, 305-311} and the recombinant peptide expressed in Escherichia coli {Fusi et al . (1995) Gene 154, 99-103} were able to hydrolyze tRNA(fMet) . However, the synthetic SSR(1-62) exhibited DNA-binding activity . In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion . Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme . These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding . Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.

Virology, 2000 Mar 1, 268(1), 218 - 25
A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid; Navarro JA et al.; Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast . Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases . On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids . The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively . To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure . High concentrations of alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) was a 25% inhibition observed . These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded .

Virology, 2000 Mar 1, 268(1), 201 - 17
Dihydrofolate reductase from Kaposi's sarcoma-associated herpesvirus; Cinquina CC et al.; Kaposi's sarcoma-associated herpesvirus (KSHV) is the first human virus known to encode dihydrofolate reductase (DHFR), an enzyme required for nucleotide and methionine biosynthesis . We have studied the purified KSHV-DHFR enzyme in vitro and analyzed its expression in cultured B-cell lines derived from primary effusion lymphoma (PEL), an AIDS-associated malignancy . The amino acid sequence of KSHV-DHFR is most similar to human DHFR (hDHFR), but the viral enzyme contains an additional 23 amino acids at the carboxyl-terminus . The viral DHFR, overexpressed and purified from E . coli, was catalytically active in vitro . The K(m) of KSHV-DHFR for dihydrofolate (FH(2)) was 2.4 microM, which is significantly higher than the K(m) of recombinant hDHFR (rhDHFR) for FH(2) (390 nM) . K(m) values for NADPH were similar for the two enzymes, about 1 microM . KSHV-DHFR was inhibited by folate antagonists such as methotrexate (K(i): 200 pM), aminopterin (K(i): 610 pM), pyrimethamine (K(i): 29 nM), trimethoprim (K(i): 2.3 microM), and piritrexim (K(i): 3.9 nM) . In all cases, K(i) values for these folate antagonists were higher for KSHV-DHFR than for rhDHFR . The viral enzyme was expressed at levels two- to tenfold higher than hDHFR in PEL cell lines as an early lytic cycle gene . KSHV-DHFR mRNA and protein appeared from 6 to 24 h after chemical induction of the KSHV lytic cycle . Epitope-tagged KSHV-DHFR and rhDHFR both localized to the nucleus of transfected cells, while other KSHV nucleotide metabolism genes localized to the cytoplasm . DHFR activity was not essential for viral replication in cultured PEL cells . Since hDHFR was not detectable in peripheral blood mononuclear cells (PBMCs), KSHV-DHFR may function to provide increased DHFR activity in vivo in infected cells that have little or none of their own enzyme .

Virology, 2000 Mar 1, 268(1), 104 - 11
Deletion mapping of the potyviral helper component-proteinase reveals two regions involved in RNA binding; Urcuqui-Inchima S et al.; The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA . To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coli expression vector . The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity . The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding . They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro . Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes . The possibility that domain B consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins .

Virology, 2000 Mar 1, 268(1), 79 - 86
Construction of a selectable nef-defective live-attenuated human immunodeficiency virus expressing Escherichia coli gpt gene; Tanuri A et al.; We have developed a replication-competent human immunodeficiency virus (HIV) carrying a selective marker that can be used in vivo . This recombinant virus (Z6 Delta nef gpt) was generated by replacing the 5' half of the HIV nef gene with the Escherichia coli guanine phosphoribosyl transferase gene (gpt) . This new vector can express the gpt product on infection and works as a positive selective marker for mycophenolic acid (MPA) resistance, a potent immunosuppressive drug used in organ rejection therapy . Conversely, gpt expression also served as a negative selectable marker, since its intracellular expression induces host-cell susceptibility to 6-thioxantine (6-TX), a nucleotide analog that is toxic to the infected cell under these conditions . In this manner, we could suppress the recombinant virus replication through 6-TX selection in both transformed cells and primary human peripheral blood mononuclear cells (PBMCs), suggesting the vector's potential as a model for a new live-attenuated vaccine approach against HIV .

Arch Biochem Biophys, 2000 Mar 1, 375(1), 171 - 4
Removal of the tryptophan 139 side chain in Escherichia coli D-3-phosphoglycerate dehydrogenase produces a dimeric enzyme without cooperative effects; Grant GA et al.; Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) is a homotetrameric enzyme whose activity is allosterically regulated by l-serine, the end-product of its metabolic pathway . Previous studies have shown that PGDH displays two modes of cooperative interaction . One is between the l-serine binding sites and the other is between the l-serine binding sites and the active sites . Tryptophan 139 participates in an intersubunit contact near the active site catalytic residues . Site-specific mutagenesis of tryptophan 139 to glycine results in the dissociation of the tetramer to a pair of dimers and in the loss of cooperativity in serine binding and between serine binding and inhibition . The results suggest that the magnitude of inhibition of activity at a particular active site is primarily dependent on serine binding to that subunit but that activity can be modulated in a cooperative manner by interaction with adjacent subunits . The disruption of the nucleotide domain interface in PGDH by mutating Trp-139 suggests the potential for a critical role of this interface in the cooperative allosteric processes in the native tetrameric enzyme .

Arch Biochem Biophys, 2000 Mar 1, 375(1), 131 - 7
Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase; Kerr KM et al.; Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH . Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+) . K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+) . This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD . Thus monovalent cation activation is linked to the NAD site . K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD . Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala . In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested . Thus these mutations eliminate cation selectivity . Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH . Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+) . However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD . Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site . Alternatively, this mutation could uncover a second monovalent cation binding site .

Arch Biochem Biophys, 2000 Mar 1, 375(1), 101 - 10
Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli; Merewether LA et al.; Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin . The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer . A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily . In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule . The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses . Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis . Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion . Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay . Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed .

Arch Biochem Biophys, 2000 Mar 1, 375(1), 7 - 20
Cystic fibrosis transmembrane conductance regulator: the purified NBF1+R protein interacts with the purified NBF2 domain to form a stable NBF1+R/NBF2 complex while inducing a conformational change transmitted to the C-terminal region; Lu NT et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) is known to function as a regulated chloride channel and, when genetically impaired, to cause the disease cystic fibrosis . The novel studies reported here were undertaken to gain greater molecular insight into possible interactions among CFTR's soluble domains, which include two nucleotide binding domains (NBF1 and NBF2) and a regulatory domain (R) . The NBF1+R and NBF2 regions of CFTR were highly expressed in Escherichia coli, purified to near homogeneity under denaturing conditions, and refolded . Both refolded proteins bound TNP-ATP and TNP-ADP, which could be readily replaced with ATP . Four different approaches were then used to determine whether the NBF1+R and NBF2 proteins interact . First, the purified NBF2 protein was labeled near its C-terminus with a fluorescent probe, 7-diethyl amino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) . Addition of the unlabeled NBF1+R to the CPM-labeled NBF2 caused a red-shift in lambda(max) of the CPM fluorescence, consistent with a direct interaction between the two proteins . Second, when the NBF1+R protein, the NBF2 protein, and a mixture of the two proteins were folded separately and analyzed by molecular sieve chomatography, the mixture was found to elute prior to either NBF1+R or NBF2 . Third, na-tive-PAGE gel studies revealed that the mixture of the NBF1+R and NBF2 domains migrated as a single band with an R(F) value between that of NBF1+R and NBF2 . Fourth, trypsin digestion of a mixture of the NBF1+R and NBF2 proteins occurred at a slower rate than that for the individual proteins . Finally, studies were carried out to determine whether an NBF1+R/NBF2 interaction could be demonstrated after expressing one of the two proteins in soluble, native form, thus avoiding the inclusion body, denaturation, and renaturation approach . Specifically, the NBF1+R protein was overexpressed in E . coli in fusion with glutathione-S-transferase near a thrombin cleavage site . Following binding of the GST-(NBF1+R) fusion protein to a GST Sepharose affinity column, added NBF2 was shown to bind and then to coelute with NBF1+R upon addition of glutathione or thrombin . Collectively, these experiments demonstrate that CFTR's NBF1+R region and its NBF2 domain, after folding separately as distinct units, have a strong propensity to interact and that this interaction is stable in the absence of added nucleotides or exogenously induced phosphorylation . These findings, together with the additional observation that the NBF1+R/NBF2 interaction induces a change in the C-terminus of NBF2, which resides within the C-terminal region of CFTR, may have important implications not only for the function of CFTR per se, but its interaction with other proteins .

Anal Biochem, 2000 Mar 1, 279(1), 96 - 9
A complete system for identifying inhibitors of creatine kinase B; Towler EM et al.; We have developed a complete system for discovery of lead compounds as inhibitors of creatine kinase B . In this article, we describe production and purification of the recombinant protein, conditions and features of an optimized high-throughput screening assay, and results of our implementation of the system using a diverse compound library .

J Bioenerg Biomembr, 1999 Dec, 31(6), 543 - 9
Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins; Kotaria R et al.; Experiments have been conducted to determine the oligomeric state of the mitochondrial citrate transport protein (CTP) from the yeast Saccharomyces cerevisiae . Both wild-type and cysteine-less (Cys-less) CTPs were overexpressed in E . coli and solubilized with sarkosyl . The purity of the solubilized material is approximately 75% . Upon incorporation into phospholipid vesicles, a high specific transport activity is obtained with both the wild-type and Cys-less CTPs, thereby demonstrating the structural and functional integrity of the preparations . Two independent approaches were utilized to determine native molecular weight . First, CTP molecular weight was determined via nondenaturing size-exclusion chromatography . With this methodology we obtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs, respectively . Second, charge-shift native gel electrophoresis was carried out utilizing a low concentration of the negatively charged detergent sarkosyl, which served to both impart a charge shift to the CTP and the protein standards, as well as to promote protein solubility . Via the second method, we obtained molecular weight values of 69,122 and 74,911 for the wild-type and Cys-less CTPs, respectively . Both methods clearly indicate that following solubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers . Furthermore, disulfide bonds are not required for either dimer formation or stabilization . The dimeric state of the CTP has important implications for the structural basis underlying the CTP translocation mechanism.

J Drug Target, 1999 Dec, 7(4), 269 - 83
Gene transfer into the CNS using recombinant adeno-associated virus: analysis of vector DNA forms resulting in sustained expression; Clark KR et al.; Recombinant adeno-associated virus (rAAV) vectors have shown significant promise as vehicles for in vivo gene transfer, particularly for transduction of organs composed primarily of non-dividing cells (i.e., muscle, CNS, and liver) . However, the mechanistic basis for this desirable property remains unclear . To investigate the fate of rAAV genomes in mouse brain, we stereotactically injected an rAAV vector carrying the E . coli lacZ gene into the caudate of BALB/c mice and demonstrate efficient transduction of mouse brain cells that possess cellular morphology consistent with post-mitotic neurons . We observed a significant increase in beta-galactosidase expression from 5 to 56 days after injection that paralleled the disappearance of single-stranded DNA input genomes . Analysis of in vivo viral DNA forms over time out to 5 months after inoculation revealed that rAAV genomes associated with high molecular weight mouse chromosomal DNA by 14 days after injection and persisted for the length of this study . The pattern of Southern hybridization was consistent with random viral integration in predominantly head-to-tail concatameric arrays . Importantly, we also documented an additional DNA species that appears to be a monomeric episomal circular form based on nuclease sensitivity assays . These data are the first to document the existence of multiple vector DNA forms present within the adult murine brain following direct rAAV inoculation and therefore, provide insight into the molecular events that ultimately result in long-term rAAV mediated transgene expression.

FEBS Lett, 2000 Jan 28, 466(2-3), 389 - 93
Subcellular localization and processing of the lytic transglycosylase of the conjugative plasmid R1; Bayer M et al.; Protein P19 encoded by the conjugative resistance plasmid R1, is essential for efficient conjugative DNA transfer and infection by the pilus-specific RNA phage R17 . Based on sequence homologies P19 belongs to a family of lysozyme-like virulence factors which are found in type III and type IV secretion systems . In this report we describe the processing and subcellular localization of P19 . Pulse-chase experiments were used to demonstrate the processing of P19 by the signal peptidase I of Escherichia coli . Translocation of P19 across the inner membrane was shown by gene 19-phoA fusions . Cell fractionation studies of P19 expressing cells showed the presence of P19 in the membrane compartment . P19 was solubilized with the detergent Sarkosyl indicating an inner membrane localization . Using sucrose density gradient centrifugation to separate inner and outer membranes, P19 was found in both membrane fractions . Taken together, our data suggest that mature P19 is a periplasmic protein which may be attached to the proposed membrane-spanning DNA transport complex.

FEBS Lett, 2000 Jan 28, 466(2-3), 372 - 6
Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase; Hewitson KS et al.; Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme . These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity . Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine . All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a {2Fe-2S}2+ cluster similar to that of the wild-type enzyme.

FEBS Lett, 2000 Jan 28, 466(2-3), 317 - 22
Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants; Stafford SJ et al.; Protein disulfide isomerase (PDI) exhibits both an oxido-reductase and an isomerase activity on proteins containing cysteine residues . These activities arise from two active sites, both of which contain pairs of redox active cysteines . We have developed two simple in vivo assays for these activities of PDI, based on the demonstration that PDI can complement both a dsbA mutation and a dsbC mutation when expressed to the periplasm of Escherichia coli . We constructed a variety of mutants in and around the active sites of PDI and analysed them using these complementation assays . Our analysis showed that the active site amino acid residues have a major role in determining the activities exhibited by PDI, particularly the N-terminal cysteine of the N-terminal active site . The roles of the histidine residue at position 38 and the glutamic acid residue at position 30 were also studied using these assays . The results show that these two in vivo assays should be useful for rapid screening of mutants in PDI prior to purification and detailed biochemical analysis.

Wei Sheng Yan Jiu, 1998 Mar, 27(2), 103 - 4, 108
{A rapid simple and reliable method for sequencing the targeted gene of shuttle vector pSP189}; Tan X et al.; The authors established a simple, rapid and effective procedures for sequencing the targeted a-gene SupF TRNA of shuttle vector pSP189, which is very important and useful in molecular mechanism research of mutagens . In this method, double-stranded plasmid DNA as template, prepared single-stranded DNA with alkali denaturation, applied r-32P-ATP for labelling 5'-end of primer, were used . A satisfactory result was achieved.

Wei Sheng Yan Jiu, 1998 Mar, 27(2), 95 - 6
{Study of ozonization effects on mineral water components}; Zhao Y et al.; The disinfection effects of ozonization and its influences on chemical components of mineral water were investigated . The results showed that ozone at the level of 0.5 mg/L and with the exposure time of 5 minutes effectively destroyed bacteria in mineral water . High level ozone showed no strong influences on some beneficial components, such as strontium and metasilicate and on some main components, such as bicarbonate, hardness and alkalinity, but slightly elevated pH value . Ozonization reduced the contents of total dissolved solids and oxygen demand, and decomposed some reductive contaminants such as ammonia, cyanide and phenols . Ozonization will convert part of the bromide into hypobromite and bromate.

Hunan Yi Ke Da Xue Xue Bao, 1998, 23(5), 425 - 8
{Schistosoma japonicum ferritin: cloning, nucleotide sequencing, expression, and purification}; Yi X et al.; Our previous work showed that immunization of mice with Schistosoma japonicum (Sj) immature eggs induced significant immunity against fecundity and embryonation of the parasite . The Sj adult cDNA library was screened by sera from rabbits against Sj immature egg antigen (RASjIEA) . The genes encoding molecules which may induce immunity against fecundity/embryonation were chosen for further cloning and expression . First of all, RASjIEA was absorbed with E . coli lysate to remove cross reactive antibodies . The cDNA library was then immunoscreened using the routine method . The resulted positive plaques were rescreened till individual clones were confirmed . Phagemids were obtained using in vivo excision . The positive clones were amplified using PCR . The sizes of the genes were determined by agarose gel electrophoresis . After DNA sequencing of the genes cloned, Gene bank was searched and six different genes were identified from a total of 102 positive clones . One of six identified genes, Sj ferritin (SjFer) was chosen to subclone into pGMC vector . According to DNA sequences of Sj Fer and MCS (multiple cloning site) of the vector, forward primer (Fer/GMC1) and reverse primer (Fer/GMC2) were designed and used to amplify Sj Fer by PCR . The Sj Fer cDNA and expression vector pGMC were digested with BamHI and XhoI . The digested cDNA and pGMC were ligased by T4 DNA ligase to construct a recombinant which was then used to transform E . coli strain ER2566 . The fusion protein GMCSF-Sj Ferritin was expressed in insoluble form, the inclusion body . Pellets were harvested and resolved in Tris-HCl buffer containing 8M urea . GMCSF-Sj Ferritin was purified by affinity chromatography using Ni-NTA resin . The molecular weight was determined by SDS-PAGE . This study first reports the gene encoding S . japonicum ferritin as a new candidate for schistosome vaccine.

Plant Cell Physiol, 1999 Dec, 40(12), 1297 - 304
Characterization of mitochondria-located small heat shock protein from tomato (Lycopersicon esculentum); Liu J et al.; We cloned and sequenced a full-length cDNA encoding the precursor of the mitochondria-located small heat shock protein (MT-sHSP) gene (LeHSP23.8) from tomato (Lycopersicon esculentum) . The deduced protein precursor with a calculated molecular weight of 23.8 kDa was predicted to target mitochondria and was classified as a plant MT-sHSP . A single copy of LeHSP23.8 was found in tomato genomic DNA by southern-blot analysis . Northern-blot analysis revealed the heat inducible character of LeHSP23.8 mRNA . The LeHSP23.8 mRNA was hardly detectable at about 36 degrees C but accumulated markedly at 40 degrees C . The molecular chaperone function of LeHSP23.8 was confirmed in vitro . The recombinant LeHSP23.8 was able to enhance the renaturation of chemically denatured citrate synthase (CS) . Moreover, the recombinant LeHSP23.8 protected CS from thermal inactivation and also promoted the renaturation of thermally inactivated citrate synthase.

Plant Cell Physiol, 1999 Dec, 40(12), 1262 - 70
Identification and expression of cotton (Gossypium hirsutum L.) plastidial carbonic anhydrase; Hoang CV et al.; Four carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark-grown cotton (Gossypium hirsutum L.) seedling cDNA library . Nucleotide sequence analysis revealed two different CA isoforms designated GhCA1 and GhCA2 . The encoded polypeptides possess N-terminal serine/threonine-rich regions indicative of plastid transit peptides, and approximately 80% sequence identity to other plant plastidial beta-CAs . The GhCA1 cDNA encodes a nearly complete preprotein of 323 amino acids with a molecular mass of 34.9 kDa and a predicted mature protein of 224 amino acids with a molecular mass of 24.3 kDa . Eleven nucleotide differences within ORFs of GhCA1 and GhCA2 result in 5 conservative amino acid substitutions . The 3' GhCA2 untranslated region contains five additional substitutions and one single nucleotide addition . GhCA1 clones, nearly full-length or with 70% of the transit peptide deleted, were expressed as LacZ alpha fusion proteins in E . coli . Lysates of these strains contained 9-fold higher levels of CA activity as compared to untransformed controls and this activity was inhibited by CA-specific inhibitors . Sulfanilamide, acetazolamide, ethoxyzolamide, each at 10 mM, inhibited recombinant CA activity approximately 50%, 65%, and 75%, respectively . In plant tissue homogenates these inhibitors reduced CA activity by 50%, 70%, and 95%, respectively . Although CA activity was bighest in extracts of mature cotton leaves, probing total RNA with GhCA1 revealed CA transcript levels to be highest in the cotyledons of dark-grown cotton seedlings . Collectively, our data indicate the presence of a plastid-localized CA in cotyledons of germinated seeds, suggesting a role for CA in postgerminative growth.

J Biotechnol, 2000 Feb 17, 77(2-3), 169 - 78
Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I; Cho WK et al.; An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I . The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction . The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system . The cloned scFv was expressed in E . coli as inclusion bodies . After purification from E . coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography . The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA) . The affinity constant was determined by a biosensor method using the BIAcore system . The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E . coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd) . A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was . This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.

Antivir Ther, 1999, 4(2), 87 - 94
In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA; Wainberg MA et al.; 9-(2-phosphonomethoxypropyl)adenine (PMPA) has demonstrated remarkable anti-simian immunodeficiency virus (SIV) activity in macaque models of SIV infection and transmission prevention . Recently, PMPA and its oral prodrug, bis-POC PMPA, have also shown potent anti-human immunodeficiency virus type 1 (HIV-1) activity in Phase I clinical studies . In vitro experiments were performed to address the resistance properties of PMPA . After eight passages in increasing concentrations of PMPA, HIV-1IIIB was able to grow in the presence of 2 microM PMPA, fivefold above the IC50 of PMPA for wild-type parental virus . Sequence analysis of the reverse transcriptase (RT) genes from four of 15 RT clones demonstrated the presence of a K65R substitution in RT and recombinant HIV expressing the K65R RT mutation showed a threefold to fourfold increase in IC50 value for PMPA as compared to wild-type . Additional experiments demonstrated that viruses expressing other nucleoside-associated RT resistance mutations all showed wild-type or < threefold reduced susceptibility to PMPA in vitro . Interestingly, lamivudine-resistant viruses expressing the M184V RT mutation showed wild-type to slightly increased susceptibility to PMPA in vitro and addition of the M184V mutation to HIV with the K65R mutation resulted in reversion to wild-type susceptibility for PMPA . In agreement with the cell culture findings, Escherichia coli-expressed K65R RT showed fivefold reduced susceptibility to PMPA diphosphate, the active moiety of PMPA . Furthermore, in combination experiments, PMPA with hydroxyurea showed synergistic inhibition of HIV replication in vitro . The potent antiretroviral activity and favourable resistance profile of PMPA and bis-POC PMPA are being further investigated in ongoing clinical trials.

J Biol Chem, 2000 Feb 25, 275(8), 5880 - 7
A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein; Aslani A et al.; The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene . It is required for the activation of the viral origin of DNA replication oriS . Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS* . Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop . Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure . OBP forms a stable complex with oriS* . We have identified structural features of oriS* recognized by OBP . The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation . Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex . We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.

J Biol Chem, 2000 Feb 25, 275(8), 5606 - 12
Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase; Soundar S et al.; Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases . The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E . coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding . The four arginines were separately mutated to glutamine using a polymerase chain reaction method . Wild type and mutant enzymes were each expressed in E . coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase . The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function . The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme . The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type . The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants . It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction . The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis . The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes . In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization . These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E . coli enzyme.

J Biol Chem, 2000 Feb 25, 275(8), 5409 - 15
Mammalian 5'(3')-deoxyribonucleotidase, cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells; Rampazzo C et al.; 5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known . It was earlier purified to homogeneity from human placenta . We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa . The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells . The recombinant enzyme was purified to homogeneity from transformed E . coli and was found to be identical with the native enzyme . After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity . A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme . Several polymerase chain reaction-based approaches to find this sequence met with no success . A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

J Biol Chem, 2000 Feb 25, 275(8), 5323 - 8
5'-nicked apurinic/apyrimidinic sites are resistant to beta-elimination by beta-polymerase and are persistent in human cultured cells after oxidative stress; Nakamura J et al.; Genomic DNA is continuously exposed to oxidative stress . Whereas reactive oxygen species (ROS) preferentially react with bases in DNA, free radicals also abstract hydrogen atoms from deoxyribose, resulting in the formation of apurinic/apyrimidinic (AP) sites and strand breaks . We recently reported high steady-state levels of AP sites in rat tissues and human liver DNA (Nakamura, J., and Swenberg, J . A . (1999) Cancer Res . 59, 2522-2526) . These AP sites were predominantly cleaved 5' to the lesion . We hypothesized that these endogenous AP sites were derived from oxidative stress . In this investigation, AP sites induced by ROS were quantitated and characterized . A combination of H(2)O(2) and FeSO(4) induced significant numbers of AP sites in calf thymus DNA, which were predominantly cleaved 5' to the AP sites (75% of total aldehydic AP sites) . An increase in the number of 5'-AP sites was also detected in human cultured cells exposed to H(2)O(2), and these 5'-AP sites were persistent during the post-exposure period . beta-Elimination by DNA beta-polymerase efficiently excised 5'-regular AP sites, but not 5'-AP sites, in DNA from cells exposed to H(2)O(2) . These results suggest that 5'-oxidized AP sites induced by ROS are not efficiently repaired by the mammalian short patch base excision repair pathway.

J Biol Chem, 2000 Feb 25, 275(8), 5318 - 22
The acidic triad conserved in type IA DNA topoisomerases is required for binding of Mg(II) and subsequent conformational change; Zhu CX et al.; The acidic residues Asp-111, Asp-113, and Glu-115 of Escherichia coli DNA topoisomerase I are located near the active site Tyr-319 and are conserved in type IA topoisomerase sequences with counterparts in type IIA DNA topoisomerases . Their exact functional roles in catalysis have not been clearly defined . Mutant enzymes with two or more of these residues converted to alanines were found to have >90% loss of activity in the relaxation assay with 6 mM Mg(II) present . Mg(II) concentrations (15-20 mM) inhibitory for the wild type enzyme are needed by these double mutants for maximal relaxation activity . The triple mutant D111A/D113A/E115A had no detectable relaxation activity . Mg(II) binding to wild type enzyme resulted in an altered conformation detectable by Glu-C proteolytic digestion . This conformational change was not observed for the triple mutant or for the double mutant D111A/D113A . Direct measurement of Mg(II) bound showed the loss of 1-2 Mg(II) ions for each enzyme molecule due to the mutations . These results demonstrate a functional role for these acidic residues in the binding of Mg(II) to induce the conformational change required for the relaxation of supercoiled DNA by the enzyme.

J Biol Chem, 2000 Feb 25, 275(8), 5270 - 4
Proximity of periplasmic loops in the metal-Tetracycline/H(+) antiporter of Escherichia coli observed on site-directed chemical cross-linking; Kubo Y et al.; Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A . (1999) FEBS Lett . 457, 169-173) . To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline . The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control . Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed . Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6 . In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking . The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.

J Biol Chem, 2000 Feb 25, 275(8), 5264 - 9
An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli; Yerushalmi H et al.; EmrE is an Escherichia coli 12-kDa protein that confers resistance to toxic compounds, by actively removing them in exchange with protons . The protein includes eight charged residues . Seven of these residues are located in the hydrophilic loops and can be replaced with either Cys or another amino acid bearing the same charge, without impairing transport activity . Glu-14 is the only charged residue in the membrane domain and is conserved in all the proteins of the family . We show here that this residue is the site of action of dicyclohexylcarbodiimide, a carbodiimide known to act in hydrophobic environments . When Glu-14 was replaced with either Cys or Asp, resistance was abolished . Whereas the E14C mutant displays no transport activity, the E14D protein shows efflux and exchange at rates about 30-50% that of the wild type . The maximal DeltapH-driven uptake rate of E14D is only 10% that of the wild type . The mutant shows a different pH profile in all the transport modes . Our results support the notion that Glu-14 is an essential part of a binding domain shared by substrates and protons but mutually exclusive in time . This notion provides the molecular basis for the obligatory exchange catalyzed by EmrE.

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2527 - 32
Protein folding and unfolding on a complex energy landscape; Leeson DT et al.; Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa . Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions . The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism . However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways . We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2046 - 51
The accuracy of codon recognition by polypeptide release factors; Freistroffer DV et al.; The precision with which individual termination codons in mRNA are recognized by protein release factors (RFs) has been measured and compared with the decoding of sense codons by tRNA . An Escherichia coli system for protein synthesis in vitro with purified components was used to study the accuracy of termination by RF1 and RF2 in the presence or absence of RF3 . The efficiency of factor-dependent termination at all sense codons differing from any of the three stop codons by a single mutation was measured and compared with the efficiency of termination at the three stop codons . RF1 and RF2 discriminate against sense codons related to stop codons by between 3 and more than 6 orders of magnitude . This high level of accuracy is obtained without energy-driven error correction (proofreading), in contrast to codon-dependent aminoacyl-tRNA recognition by ribosomes . Two codons, UAU and UGG, stand out as hotspots for RF-dependent premature termination.

J Drug Target, 1999, 7(3), 223 - 32
Characterization of norfloxacine release from tablet coated with a new pH-sensitive polymer, P-4135F; Hu Z et al.; A new pH-sensitive polymer, P-4135F, was evaluated as a colon delivery device for norfloxacine (NFLX) which is used for the therapy of patients with Vero toxin-producing Escherichia coli gastroenteritis . P-4135F has a dissolution threshold pH of 7.2 which is higher than the conventional pH-sensitive polymers, Eudragit S100 and L100 . To compare the dissolution site of P-4135F coated tablets with other enteric polymer coatings, mini-tablets containing sodium fluorescein (FL) as a model drug were prepared by coating them with the three polymers . After oral administration of FL mini-tablets to rats, the first-appearance time, Ti, of FL into the systemic circulation was measured . The Tis were 0.7+/-0.2 h for Eudragit L100, 1.8+/-0.4 h for S100 and 2.0+/-0.3 h for P-4135F . Direct inspection of the dissolution process of the FL mini-tablets after oral administration to rats was performed by abdominal incision studies . All of the coated FL mini-tablets started to dissolve in the rat ileum . The dissolution sites were identified to be proximal to the ileocecal junction for P-4135F, at the middle part of the ileum for Eudragit S100 and at the proximal part of the ileum for Eudragit L100 . NFLX tablets with different membrane thicknesses of P-4135F were prepared and were orally administered to beagle dogs . The colon delivery efficiency was evaluated by measuring the Ti of NFLX into the systemic circulation . The mean Tis were 1.33+/-0.33 h for 56.8+/-0.5 microm membranes, 3.75+/-0.25 h for 64.6+/-0.7 microm membranes, 4.00+/-1.00 h for 70.5+/-0.5 microm membranes and 3.00+/-1.00 h for 74.9+/-0.4 microm membranes . By comparing the Ti, 4.33+/-0.33 h, obtained after oral administration of NFLX in a pressure-controlled colon delivery capsule, and the colon arrival time, 3.5+/-0.3 h, determined by a sulfasalazine test in beagle dogs . P-4135F coated NFLX tablets appeared to dissolve and disintegrate before reaching the colon . Studies using rats and beagle dogs have suggested that P-4135F dissolves in the lower part of the small intestine, i.e., the ileum . These studies also suggest that this new polymer will be useful for the delivery of NFLX to the lower part of the small intestine.

J Virol Methods, 2000 Feb, 84(2), 117 - 26
A simple purification and fluorescent assay method of the poliovirus 3C protease searching for specific inhibitors; Hata S et al.; Picornaviruses such as poliovirus, foot-and-mouth disease virus, and encephalomyocarditis virus produce their proteins by translating their genomic RNA, injected within the host cell, into a precursor polyprotein, which is then subjected to precise processing . The polyprotein is cleaved into mature proteins predominantly by the viral 3C protease . A simple purification and assay method for poliovirus 3C protease for use for screening for inhibitors of the 3C protease is described . A poliovirus cDNA fragment containing the 3C protease coding region was inserted into pET22b vector and expressed in Escherichia coli . The His-tagged protein (3CD'-His) was purified by a Ni-affinity column and the activity of the purified enzyme was measured by a fluorescent assay with a fluorogenic substrate containing the 3C-specific cleavage site, MocAc-MEALFQGPLQY-Dnp . The kinetic parameters calculated from the Lineweaver-Burk plot and the effects of inhibitors showed that E . coli expression with His tag and the assay using the fluorogenic substrate are efficient, simple and sensitive methods for purifying the 3C protease, and measuring its activity.

Can J Vet Res, 2000 Jan, 64(1), 15 - 20
Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil; Aidar L et al.; One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of Sao Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene . Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin . Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein . Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable . The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively . The other strains did not adhere to HEp-2 cells . To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E . coli among bovine diarrheogenic E . coli . It is also the first report from Brazil demonstrating the presence of bovine E . coli harboring the eae gene.

Cancer Lett, 2000 Jan 1, 148(1), 81 - 6
Trans-4-hydroxy-2-nonenal, an aldehydic lipid peroxidation product, lacks genotoxicity in lacI transgenic mice; Nishikawa A et al.; In order to cast light on the significance of lipid peroxidation products for carcinogenesis, the lacI mutant frequency (MF), micronucleus induction and cell proliferation were analyzed in lacI transgenic mice treated with trans-4-hydroxy-2-nonenal (HNE), a typical example . Male mice were ip injected with HNE at doses of 0, 5 or 50 mg/kg bw and 48 h thereafter, peripheral blood was collected for analyzing micronucleus induction . After 14 days, the mice were sacrificed to allow tissue sampling for examination of lacI MF and cell proliferative activity . Sixty percent of the mice given 50 mg/kg HNE died within 5 days after the treatment, but no other mortalities were observed . Histopathologically, marked pulmonary hemorrhage was found in the 50 mg/kg HNE group mice that survived until day 14 . Immunohistochemically, HNE-modified proteins were detected in their alveolar macrophages . The HNE treatment did not increase lacI MF in the liver, kidney and lung and no significant increase in micronucleus induction or cell proliferation in major organs was found in either treatment . Moreover, no tumors developed in the 5 mg/kg HNE-treated mice which survived until week 78 . Our results thus indicate that HNE lacks in vivo genotoxicity in lacI transgenic mice even when lethal doses are applied.

Gene Ther, 2000 Jan, 7(1), 80 - 7
New tools for the generation of E1- and/or E3-substituted adenoviral vectors; Danthinne X et al.; We have designed new vectors for the construction of recombinant adenoviruses containing expression cassettes in the E1 and/or E3 regions . Using a versatile set of restriction enzymes, the cassettes are cloned into small bacterial vectors and subsequently introduced into large plasmids containing the adenoviral sequences . Two positive selection markers facilitate the recovery of a cosmid containing a copy of the sequence of the recombinant adenovirus . The resulting cosmid is transfected into 293 or 911 cells in order to rescue the virus . Importantly, the method does not require any recombination event, either in E . coli or in mammalian cells . The entire procedure can generate viral plaques in 12 days . Gene Therapy (2000) 7, 80-87.

Curr Opin Struct Biol, 2000 Feb, 10(1), 87 - 94
Single-stranded-RNA binding proteins; Antson AA; Our knowledge of protein interactions with RNA molecules has been, so far, largely restricted to cases in which the RNA itself is folded into a secondary and/or tertiary structure stabilised by intramolecular base pairing and stacking . Until recently, only limited structural information has been available about protein interactions with single-stranded RNA . A breakthrough in our understanding of these interactions came in 1999, with the determination of four crystal structures of protein complexes with extended single-stranded RNA molecules . These structures revealed wonderfully satisfying patterns of the ability of proteins to accommodate RNA bases, with the sugar-phosphate backbone often adopting conformations that are different from the classical double helix.

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 909 - 15
In vitro binding of nucleolin to double-stranded telomeric DNA; Pollice A et al.; We have purified a 100 kDa protein, resolved in a Southwestern binding screen of total nuclear proteins from Hela cells with double-stranded human telomeric probe . A polyclonal antiserum raised by this protein recognizes purified nucleolin and stains nucleoli in growing Hela cells . We demonstrate that a truncated form of human nucleolin and a purified deletion derivative of mouse nucleolin bind in vitro to duplex telomeric DNA . This study suggests a new link between telomeres and the nucleolus .

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 740 - 4
Clay-bridged electron transfer between cytochrome p450(cam) and electrode; Lei C et al.; We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite . The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process . The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively . These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam) . The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution . UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution . However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential .

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 724 - 7
3-Methyladenine-DNA glycosylase I from Escherichia coli-computer modeling and supporting experimental evidence; Plochocka D et al.; TagA (3-methyladenine-DNA glycosylase I) excises 3-methyadenine and 3-methylguanine from alkylated DNA . The structure of this enzyme has not yet been determined experimentally . We propose a three-dimensional model of the TagA protein based on the threading algorithm . The model shows that TagA is a mostly alpha-helical protein, in agreement with circular dichroism measurements . None of the eight cysteines present in the TagA sequence forms a disulfide bridge in the model structure, which has also been experimentally verified with the use of Ellman method .

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 692 - 6
Human glial cell-line-derived neurotrophic factor: a structure-function analysis; Chen ZY et al.; Glial cell-line-derived neurotrophic factor (GDNF) is a protein known to enhance the survival of dopaminergic and motor neurons . It has been shown to have therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases . GDNF gene was modified by deletion and insertion mutagenesis using PCR methods . The various mutants were all highly expressed in Escherichia coli . The recombinant proteins were purified and their survival-promoting activities were determined by motor neurons . The result showed that the C-terminus was critical for structure stability of GDNF, and the alpha-helix, finger1 and finger2 regions were involved in receptor binding, while the N-terminus was not essential for the biological functions of GDNF .

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 553 - 61
Growth suppression of Escherichia coli by induction of expression of mammalian genes with transmembrane or ATPase domains; Inoue S et al.; Growth inhibition of Escherichia coli host cells is frequently observed when some mammalian genes are induced to express exogenously . To find common features of these mammalian genes, an assay was designed for the isolation of these genes which show growth-inhibitory effect on E . coli by induction of expression . Of 38,000 clones derived from a mouse brain cDNA library, 64 cDNA clones were systematically selected out by this method, of which 45 clones had putative open reading frames encoding proteins with putative membrane-associated regions or ATP-binding/ATPase activities . These results show that a fraction of membrane-associated proteins or ATP-binding/ATPase genes can be isolated from cDNA libraries by our simple method .

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 390 - 4
Light chain of natural antibody plays a dominant role in protein antigen binding; Song MK et al.; Examinations of the contribution and the specificity of heavy (H) and light (L) chains of natural antibodies to antigen binding may help us to better understand antigen recognition and the development of naive B cells . We previously generated natural Fab antibody fragments reactive to preS1 of HBV using a naive, non-immunized Fab antibody library derived from peripheral B cells of a normal healthy volunteer . We now constructed expression vectors for the Fd (VH + CH1), L chain, and scFv fragments using the sequences encoding parental Fabs as a source of natural antibody genes . The recombinant antibody fragments were expressed as inclusion bodies in E . coli BL21 (DE) cells . When denatured and then refolded, the antibody fragments retained their binding properties . Recombinant L chains and scFvs exhibited three- to 40-fold higher affinities (in the order of 10(7) M(-1)) over the parental Fabs, whereas the affinities of Fds (in the order of 10(5) M(-1)) were much lower compared to the parental Fabs . The results obtained from sandwich ELISA revealed that the L chains bound the virus more efficiently than Fds . Additional experiments were performed to evaluate the specificity of the recombinant fragments for surface proteins of HBV . Fds and L chains were reactive towards HBsAg and the preS2 peptide as well as preS1 and showed patterns of epitope recognition quite different from those of parental Fabs . The data presented here demonstrate that the prominence of the L chain in determining protein binding activity is a property of natural antibodies and is quite unlike the antibodies induced by immunization, and that the specificity of Fab is not determined by the individual antibody chain but by the correct pairing of H and L chain .

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 298 - 301
Cloning and characterization of a putative human d-2-hydroxyacid dehydrogenase in chromosome 9q; Huang T et al.; There is little information on d-isomer-specific dehydrogenases in humans . Identification of d-2-hydroxyglutaric aciduria, an inherited metabolic disorder associated with severe neurological dysfunction, highlights the role of d-isomers in human metabolism . The possibility of a defect in d-2-hydroxyglutarate dehydrogenation prompted us to employ E . coli d-2-hydroxyacid dehydrogenase cDNA to search the human expressed sequence tags database . Two human EST homologues were retrieved and sequenced . Analysis showed the two clones were identical with 1258 nucleotides encoding 248 amino acids of the putative human d-2-hydroxyacid dehydrogenase . It was highly homologous to bacterial d-2-hydroxyacid dehydrogenases (46%), d-phosphoglycerate dehydrogenase (38%), and formate dehydrogenase (36%) at the amino acid level . The gene is expressed ubiquitously in tissue, most abundantly in liver, and was mapped to chromosome 9q between markers WI-3028 and WI-93330 . To our knowledge this is the first cloning and characterization of the cDNA for a human d-isomer specific NAD(+)-dependent 2-hydroxyacid dehydrogenase .

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 282 - 8
Isolation and characterization of dcw cluster from Streptomyces collinus producing kirromycin; Mikulik K et al.; A 4.5-kb BamHI fragment of chromosomal DNA of Streptomyces collinus containing gene ftsZ was cloned and sequenced . Upstream of ftsZ are localized genes ftsQ, murG, and ftsW, and downstream is yfiH . Gene ftsA is not adjacent to ftsZ or other genes of the cloned fragment . Protein FtsZ was isolated and characterized with respect to its binding to GTP and GTPase activity . The binding of GTP to FtsZ was Ca(2+) or Mg(2+) dependent with an optimum at 10 mM . The rate of GTP hydrolysis by FtsZ was stimulated by KCl . The presence of Ca(2+) (3-5 mM) resulted in a significant increase of GTPase activity . Higher concentrations of Ca(2+) than 5 mM had an inhibitory effect on GTPase activity . These results indicate that divalent ions (Ca(2+) or Mg(2+)) can be involved in regulation of GTP binding and hydrolysis of FtsZ . The maximum level of FtsZ was detected in aerial mycelium when spiral loops and sporulation septa were formed . FtsZ is degraded after finishing sporulation septa .

Antimicrob Agents Chemother, 2000 Mar, 44(3), 647 - 50
Characterization of the fomA and fomB gene products from Streptomyces wedmorensis, which confer fosfomycin resistance on Escherichia coli; Kobayashi S et al.; Together, the fomA and fomB genes in the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis confer high-level fosfomycin resistance on Escherichia coli . To elucidate their functions, the fomA and fomB genes were overexpressed in E . coli and the gene products were characterized . The recombinant FomA protein converted fosfomycin to fosfomycin monophosphate, which was inactive on E . coli, in the presence of a magnesium ion and ATP . On the other hand, the recombinant FomB protein did not inactivate fosfomycin . However, a reaction mixture containing FomA and FomB proteins converted fosfomycin to fosfomycin monophosphate and fosfomycin diphosphate in the presence of ATP and a magnesium ion, indicating that FomA and FomB catalyzed phosphorylations of fosfomycin and fosfomycin monophosphate, respectively . These results suggest that the self-resistance mechanism of the fosfomycin-producing organism S . wedmorensis is mono- and diphosphorylation of the phosphonate function of fosfomycin catalyzed by FomA and FomB.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 225 - 35
Refolding and purification of a urokinase plasminogen activator fragment by chromatography; Fahey EM et al.; A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E . coli in the form of inclusion bodies . Purification and renaturation was achieved in a three-stage process . Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation . Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS) . Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of Mr 10000-1500000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant . Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA . The result is concentrated, active, homogeneous u-PA.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 213 - 24
Expression of chymotrypsin(ogen) in the thioredoxin reductase deficient mutant strain of Escherichia coli AD494(DE3) and purification via a fusion product with a hexahistidine-tail; Verheyden G et al.; A reliable protocol was designed for fast expression and purification of recombinant chymotrypsin(ogen) . The zymogen was overexpressed in soluble form as a (His)6-fusion construct in the cytoplasm of the thioredoxin reductase deficient Escherichia coli strain AD494(DE3) . This allowed purification of chymotrypsinogen in a highly selective affinity chromatography capture step using a Ni-NTA column . After activation with enterokinase, the enzymatically active chymotrypsin was purified in a polishing step using a modified soybean trypsin inhibitor agarose column . This expression system and the use of affinity chromatography for capture and polishing, offers an easier and faster route to recombinant chymotrypsin(ogen) than the previously described use of Saccharomyces cerevisiae.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 195 - 202
D-Xylose metabolism by Candida intermedia: isolation and characterisation of two forms of aldose reductase with different coenzyme specificities; Mayr P et al.; To study individual enzyme components responsible for the initial step of D-xylose utilisation by the yeast Candida intermedia, a two-step protocol has been developed that enables clear-cut separation and isolation of two structurally similar but functionally different aldose reductases (ALRs) in high yield . In the first step, the yeast cell extract is fractionated efficiently by biomimetic chromatography using the dye HE-3B (reactive Red 120) as pseudoaffinity ligand coupled to Sepharose CL-4B . In the second step, optimised high-resolution anion-exchange chromatography using Mono Q yields purified ALR1 and ALR2 in overall yields of 63 and 62%, respectively . ALR1 is strictly specific for NADPH (2.4 x 10(5) M(-1) s(-1)) whereas ALR2 utilises NADH and NADPH with similar specificity constants of approximately 2-4 x 10(5) M(-1) s(-1) . Both enzymes are dimers with a subunit molecular mass of 36000 but they differ in pI and the number of titratable sulphydryl groups in the native protein . The chromatographic procedure identifies microheterogeneity in recombinant aldose reductase from Candida tenuis overexpressed in Escherichia coli.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 187 - 94
Purification of a D-hydantoinase using a laboratory-scale streamline phenyl column as the initial step; Abendroth J et al.; A D-hydantoinase from Thermus sp . was overexpressed in Escherichia coli and purified to homogeneity for subsequent crystallization . The purification was performed with hydrophobic interaction chromatography as the capture step followed by anion-exchange chromatography and gel permeation chromatography as intermediate purification and polishing steps, respectively . The hydrophobic interaction step was done in fluidized bed mode in a laboratory-scale Streamline column made from conventional laboratory equipment . The whole purification protocol could be finished within one day . The purified enzyme crystallizes . The crystals are suitable for X-ray protein structure analysis and diffract to at least 2.3 A resolution . Complete data sets have been measured up to 2.6 A resolution . The X-ray structure is currently being solved.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 179 - 86
Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins; Pietzsch M et al.; Two enzymes, hydantoinase (HyuH) and L-N-carbamoylase (HyuC), are required for the biocatalytic production of natural and unnatural, optically pure L-amino acids starting from D,L-5-monosubstituted hydantoins using the so called 'hydantoinase-method' . For the preparation of immobilized enzymes, which omit several drawbacks of whole cell biocatalysts, purified or at least enriched HyuH and HyuC have to be provided . In order to simplify existing purification protocols several genetically modified derivatives of HyuH and HyuC from Arthrobacter aurescens DSM 3747 have been cloned and expressed in E . coli . A fusion protein consisting of maltose-binding protein (MalE) and HyuH resulted in an enhanced solubility of the hydantoinase, which easily forms inclusion bodies . On the other hand the fusion protein could easily be purified with high yield (76%) by just one chromatographic step (amylose resin) and the complex purification protocol of the wild-type enzyme could therefore be simplified and shortened significantly . Interestingly, the specific activity of the MalE-HyuH fusion protein was as high as the wild-type enzyme despite that the molecular mass was doubled . A second modification of HyuH carrying a histidine-tag was efficiently bound to a metal affinity matrix but inactivated completely during elution from the column at either low pH or in the presence of imidazole . In the case of HyuC, an aspartate-tag has been added to the biocatalyst to allow an integrated purification-immobilization procedure since this enzyme is immobilized efficiently only via its carboxylic groups . The diminished isoelectric point of the Asp-tagged HyuC resulted in a simplified purification procedure . Compared to the wild-type enzyme expressed in E . coli HyuC-Asp6 was shifted off the elution range of the contaminating proteins and higher purification factors were obtained even in the capturing step . In contrast to HyuH, it was possible to purify a L-N-carbamoylase carrying a histidine-tag to apparent homogeneity using immobilized metal affinity chromatography . Therefore, the existing three step purification protocol was reduced to one chromatographic step and the yield of this relatively unstable protein enhanced remarkably.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 85 - 95
Expression, purification and biological properties of the carboxyl half part of the HTLV-I surface envelope glycoprotein; Tallet B et al.; The carboxyl half of the surface envelope protein of HTLV-I contains the major immunodominant and neutralizable domains . Using two affinity chromatography steps and a combination of high salt concentration and non-ionic detergent, we purified this part of the envelope protein from Escherichia coli . Analysis of some immmunological and biological properties of this protein indicated that it was folded in a way that preserved the correct structure of this domain of the HTLV-I envelope protein . It could be utilized in structural studies to further understand the mechanisms of HTLV-I entry and to better define the component(s) of an effective vaccine.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 77 - 84
Purification of a viral coat protein by an engineered polyionic sequence; Stubenrauch K et al.; Virus-like particles composed of the polyoma coat protein VP1 were produced as a central building block of an artificial vector system for gene therapy . For this purpose, recombinant VP1 was expressed in E . coli . Classical purification schemes resulted only in low yields of protein . Therefore, we developed a new affinity purification procedure . We decided to use a polyionic sequence containing eight glutamic acid residues which allows efficient purification using ion-exchange chromatography . This peptide was inserted in a solvent exposed loop on the surface of VP1 . After recombinant expression and cell lysis the first purification and concentration step consisted of a fractionated ammonium sulfate precipitation . The resuspended VP1 was loaded on an anion-exchange column . Elution with ca . 600 mM NaCl yielded almost homogeneous protein . Subsequently a size exclusion chromatography was performed to separate the pentameric VP1 from higher oligomeric and aggregated material . In contrast to wildtype VP1 the highly charged mutant form showed no significant tendency to aggregate . To demonstrate the functional state of the VP1 mutant, the in vitro assembly was investigated . At conditions similar to those for wildtype VP1 assembly, the mutant protein could form homogeneous virus-like particles.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 63 - 9
Chromatographic purification of the C(H)2 domain of the monoclonal antibody MAK33; Thies MJ et al.; The C(H)2 domain, one of the constant domains of the murine monoclonal antibody MAK33 (immunoglobulin subtype K/IgG1) was expressed in Escherichia coli forming insoluble inclusion bodies (IBs) and purified by a three-step process including a denaturation-renaturation step, hydrophobic interaction and gel permeation chromatography . After disrupting the cells, the soluble protein fraction was removed by several centrifugation steps . The isolation of the IBs from the cell fragments was achieved by solubilizing the IBs with 6 M guanidinium hydrochloride (GdmCl) and 0.1 M 1,4-dithioerythrit (DTE) to reduce all disulfide bonds . After refolding the C(H)2 domain, 1.5 M (NH4)2SO4 was added to the protein solution in order to precipitate contaminations . Then the protein was loaded on a Butyl-Sepharose fast flow column and eluted with a linear gradient {1.5-0 M (NH4)2SO4} . As the last purification step a gel permeation chromatography was run on a Superdex 75 prep grade . Finally, the purity of the C(H)2 protein was determined by a silver-stained sodium dodecyl sulfate polyacrylamide gel . We achieved a typical yield of 0.5 mg pure protein per 1 g of wet cells.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 39 - 45
Purification of the Ca2+-binding protein S100A1 from myocardium and recombinant Escherichia coli; Ehlerman P et al.; S100A1 is a new regulatory protein of myocardial contractility that is differentially expressed in early and late stages of myocardial hypertrophy . In order to further investigate the multiple functions of S100A1 in various assay systems we developed a new strategy for isolating biologically active S100A1 protein . After EDTA extraction of myocardium or recombinant bacteria, S100A1 was purified by Octyl-Sepharose hydrophobic interaction chromatography and HiTrapQ anion-exchange chromatography yielding 1.4-2.0 mg/100 g wet tissue and 0.7-1.0 mg/100 ml bacterial culture . Native porcine as well as human recombinant S100A1 revealed biological activity in physiological and biochemical assays.

Curr Opin Genet Dev, 2000 Feb, 10(1), 32 - 8
RecQ family helicases: roles in cancer and aging; Karow JK et al.; The RecQ family of DNA helicases includes at least three members in humans that are defective in genetic disorders associated with cancer predisposition and/or premature aging . Recent studies have shed light on the roles of RecQ helicases in suppressing 'promiscuous' genetic recombination and in ensuring accurate chromosome segregation . In particular, the biochemical properties of several family members have been characterised and functional interactions with other nuclear proteins have been defined.

Nature, 2000 Feb 3, 403(6769), 567 - 71
Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins; Prakash B et al.; Interferon-gamma is an immunomodulatory substance that induces the expression of many genes to orchestrate a cellular response and establish the antiviral state of the cell . Among the most abundant antiviral proteins induced by interferon-gamma are guanylate-binding proteins such as GBP1 and GBP2 . These are large GTP-binding proteins of relative molecular mass 67,000 with a high-turnover GTPase activity and an antiviral effect . Here we have determined the crystal structure of full-length human GBP1 to 1.8 A resolution . The amino-terminal 278 residues constitute a modified G domain with a number of insertions compared to the canonical Ras structure, and the carboxy-terminal part is an extended helical domain with unique features . From the structure and biochemical experiments reported here, GBP1 appears to belong to the group of large GTP-binding proteins that includes Mx and dynamin, the common property of which is the ability to undergo oligomerization with a high concentration-dependent GTPase activity.

Nature, 2000 Feb 3, 403(6769), 564 - 7
Evidence for stabilizing selection in a eukaryotic enhancer element; Ludwig MZ et al.; Eukaryotic gene expression is mediated by compact cis-regulatory modules, or enhancers, which are bound by specific sets of transcription factors . The combinatorial interaction of these bound transcription factors determines time- and tissue-specific gene activation or repression . The even-skipped stripe 2 element controls the expression of the second transverse stripe of even-skipped messenger RNA in Drosophila melanogaster embryos, and is one of the best characterized eukaryotic enhancers . Although even-skipped stripe 2 expression is strongly conserved in Drosophila, the stripe 2 element itself has undergone considerable evolutionary change in its binding-site sequences and the spacing between them . We have investigated this apparent contradiction, and here we show that two chimaeric enhancers, constructed by swapping the 5' and 3' halves of the native stripe 2 elements of two species, no longer drive expression of a reporter gene in the wildtype pattern . Sequence differences between species have functional consequences, therefore, but they are masked by other co-evolved differences . On the basis of these results, we present a model for the evolution of eukaryotic regulatory sequences.

Cell, 2000 Feb 4, 100(3), 357 - 65
Covalent modification regulates ligand binding to receptor complexes in the chemosensory system of Escherichia coli; Li G et al.; In the Escherichia coli chemosensory pathway, receptor modification mediates adaptation to ligand . Evidence is presented that covalent modification influences ligand binding to receptors in complexes with CheW and the kinase CheA . Kinase inhibition was measured with serine receptor complexes in different modification levels; Ki for serine-mediated inhibition increased 10,000-fold from the lowest to the highest level . Without CheA and CheW, ligand binding is unaffected by covalent modification; thus, the influence of covalent modification is mediated only in the receptor complex, a conclusion supported by an analogy to allosteric enzymes and the observation of cooperative kinase inhibition . Also, the finding that a subsaturating serine concentration accelerates active receptor-kinase complex assembly implies that the assembly/disassembly process may also contribute to kinase regulation.

Int J Parasitol, 2000 Jan, 30(1), 51 - 8
Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells; Nishikawa Y et al.; Monoclonal antibodies were produced against Neospora caninum tachyzoites to identify antigens which may play a role during invasion of host cells . Confocal laser microscopy showed that most antigens recognised by the mAb were located on the surface, but one mAb, 1A5, reacted to the apical end of the parasite . Some mAbs, which recognised 70, 42 and 36kDa parasite proteins, significantly inhibited the invasion of the parasite in vitro . The mAbs which recognised 42 and 36kDa parasite protein, reacted with Nc-p43 and Nc-p36 expressed by vaccinia virus and Escherichia coli, respectively . These results suggest that a 70kDa protein, Nc-p43 and Nc-p36 are involved in the invasion of the parasite into host cells.

J Gen Virol, 2000 Mar, 81(Pt 3), 759 - 67
Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence; Tomei L et al.; The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein . The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus . The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E . coli . In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity . The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences . The largest difference in the polymerase activity was observed on the latter . On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme . The elongation rates of the two enzyme forms were compared under single processive cycle conditions . Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.

J Gen Virol, 2000 Mar, 81(Pt 3), 605 - 15
Nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs; Fazeli CF et al.; The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12394 nts determined . It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract . The first ORF (ORF 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein . The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon . This ORF encodes an RNA-dependent RNA polymerase of M(r) 59276 . ORF 2 encodes a small hydrophobic protein of M(r) 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an M(r) of 59500 . ORF 4 encodes a protein with an M(r) of 54648 that shows similarity to the corresponding proteins of other closteroviruses . ORF 5 encodes the viral coat protein (CP) with an M(r) of 35416 . The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody . ORFs 6 and 7 code for two CP-related products with M(r) of 55805 and 50164, respectively . ORFs 8 and 9 encode proteins of M(r) 21558 and 23771 with unknown functions . Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome . Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.

EMBO J, 2000 Feb 15, 19(4), 776 - 85
Tn10 transpososome assembly involves a folded intermediate that must be unfolded for target capture and strand transfer; Sakai JS et al.; Tn10 transposition, like all transposition reactions examined thus far, involves assembly of a stable protein-DNA transpososome, containing a pair of transposon ends, within which all chemical events occur . We report here that stable Tn10 pre-cleavage transpososomes occur in two conformations: a folded form which contains the DNA-bending factor IHF and an unfolded form which lacks IHF . Functional analysis shows that both forms undergo double strand cleavage at the transposon ends but that only the unfolded form is competent for target capture (and thus for strand transfer to target DNA) . Additional studies reveal that formation of any type of stable transpososome, folded or unfolded, requires not only IHF but also non-specific transposase-DNA contacts immediately internal to the IHF-binding site, implying the occurrence of a topo- logically closed loop at the transposon end . Overall, transpososome assembly must proceed via a folded intermediate which, however, must be unfolded in order for intermolecular transposition to occur . These and other results support key features of a recently proposed model for transpososome assembly and morphogenesis.

EMBO J, 2000 Feb 15, 19(4), 758 - 66
DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA; Hollis T et al.; The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA . The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA . The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove . The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal . Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides . The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA . Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.

EMBO J, 2000 Feb 15, 19(4), 749 - 57
Structure of Hsp15 reveals a novel RNA-binding motif; Staker BL et al.; We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome . Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif . This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA . The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site . An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif . This may imply a hitherto unrecognized functional similarity between these three protein classes.

EMBO J, 2000 Feb 15, 19(4), 741 - 8
Hsp15: a ribosome-associated heat shock protein; Korber P et al.; We are analyzing highly conserved heat shock genes of unknown or unclear function with the aim of determining their cellular role . Hsp15 has previously been shown to be an abundant nucleic acid-binding protein whose synthesis is induced massively at the RNA level upon temperature upshift . We have now identified that the in vivo target of Hsp15 action is the free 50S ribosomal subunit . Hsp15 binds with very high affinity (K(D) <5 nM) to this subunit, but only when 50S is free, not when it is part of the 70S ribosome . In addition, the binding of Hsp15 appears to correlate with a specific state of the mature, free 50S subunit, which contains bound nascent chain . This provides the first evidence for a so far unrecognized abortive event in translation . Hsp15 is suggested to be involved in the recycling of free 50S subunits that still carry a nascent chain . This gives Hsp15 a very different functional role from all other heat shock proteins and points to a new aspect of translation.

EMBO J, 2000 Feb 15, 19(4), 542 - 9
YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase; Scotti PA et al.; In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase . Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase . Here, we use a cross-linking approach to show that hydrophilic portions of a co-translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface . The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase-associated component that we identify as YidC . YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane . We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.

EMBO J, 2000 Feb 15, 19(4), 531 - 41
Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity; de Leeuw E et al.; FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane . It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear . FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain) . FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected . In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids . The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain . Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity . We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.

J Struct Biol, 2000 Feb, 129(1), 96 - 9
Crystallization and 1.1-A diffraction of chorismate lyase from Escherichia coli; Stover C et al.; Chorismate pathway enzymes are important as producers of nonnucleotide aromatic compounds . The enzyme chorismate lyase from Escherichia coli has been crystallized in four distinct forms, three of which have been characterized by X-ray diffraction . Despite widespread screening, all four crystal forms grow from the same chemical conditions . The wild-type enzyme tends to aggregate, even in the presence of reducing agent, and yielded only one crystal form (monoclinic, form 1) that grew in intricate clusters . Chemical modification of the cysteines mitigated problems with aggregation and solubility but did not affect crystal growth behavior . Protein aggregation was largely eliminated by mutating the protein's two cysteines to serines . The double mutant retains full enzymatic activity and crystallizes in three new forms, one of which (triclinic) diffracts to 1.1-A resolution .

FEMS Microbiol Lett, 2000 Feb 15, 183(2), 259 - 64
Aldolases of the DhnA family: a possible solution to the problem of pentose and hexose biosynthesis in archaea; Galperin MY et al.; Sequence analysis of the recently identified class I aldolase of Escherichia coli (dhnA gene product) helped to identify its homologs in Chlamydia trachomatis, Chlamydiophyla pneumoniae and in each of the completely sequenced archaeal genomes . Iterative database searches revealed sequence similarities between the DhnA-family enzymes, deoxyribose phosphate aldolases and bacterial (class II) fructose bisphosphate aldolases and allowed prediction of similar three-dimensional structures (TIM-barrel fold) in all these enzymes . The Schiff base-forming lysyl residues of DhnA and deoxyribose phosphate aldolase are conserved in all members of the DhnA and deoxyribose phosphate aldolase families, indicating that these enzymes share common features with both class I and class II aldolases . The DhnA-family enzymes are predicted to possess an aldolase activity and to play a critical role in sugar biosynthesis in archaea.

FEBS Lett, 2000 Feb 11, 467(2-3), 353 - 8
The monomeric polypeptide comprises the functional flavanone 3beta-hydroxylase from Petunia hybrida; Lukacin R et al.; Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthesis of flavonoids, catechins and anthocyanidins . The enzyme had been partially purified from Petunia hybrida and proposed to be active as a dimer of roughly 75 kDa in size . More recently, the Petunia 3beta-hydroxylase was cloned and shown to be encoded in a 41655 Da polypeptide . In order to characterize the molecular composition, the enzyme was expressed in a highly active state in Escherichia coli and purified to apparent homogeneity . Size exclusion chromatographies of the pure, recombinant enzyme revealed that this flavanone 3beta-hydroxylase exists in functional monomeric and oligomeric forms . Protein cross-linking experiments employing a specific homobifunctional sulfhydryl group reagent or the photochemical activation of tryptophan residues confirmed the tendency of the enzyme to aggregate to oligomeric complexes in solution . Thorough equilibrium sedimentation analyses, however, revealed a molecular mass of 39 . 2+/-12 kDa for the recombinant flavanone 3beta-hydroxylase . The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3beta-hydroxylase of P . hybrida, which may readily associate in vivo with other proteins.

FEBS Lett, 2000 Feb 11, 467(2-3), 321 - 5
A novel target of lithium therapy; Yenush L et al.; Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems . These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy . A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity . The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM) . The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate . Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM) . Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.

FEBS Lett, 2000 Feb 11, 467(2-3), 279 - 84
Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization; Asenjo A et al.; Purified human respiratory syncytial virus (HRSV) P phosphoprotein from transfected HEp-2 cells is able to oligomerize forming tetramers . The bulk of constitutive P protein phosphorylation (99 . 8%) (serine residues 116, 117, 119, 232 and 237) can be removed without affecting protein oligomerization . However, dephosphorylated P protein, produced in bacteria, is unable to oligomerize . This difference can be explained by a transient P protein phosphorylation, detected in HEp-2 cells, that could be essential for P protein oligomerization.

FEBS Lett, 2000 Feb 11, 467(2-3), 195 - 200
Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii; Matsui I et al.; A beta-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli . The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85 degrees C for 15 min . The optimum pH was 6.0 and the optimum temperature was over 100 degrees C, respectively . BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90 degrees C (pH 6.0) was 15 h . BGPh has a novel substrate specificity with k(cat)/K(m) values high enough for hydrolysis of beta-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of beta-linked glucose dimer more hydrophilic than aryl- or alkyl-beta-D-Glcp.

FEBS Lett, 2000 Feb 11, 467(2-3), 189 - 94
Drosophila MTN: a metazoan copper-thionein related to fungal forms; Valls M et al.; Two Drosophila metallothioneins (MT) have been reported: MTN, a 40 residue peptide including 10 Cys, and MTO, a 43 residue peptide including 12 Cys . However, neither functional nor evolutionary analyses for either of the Drosophila MT are available . Here, heterologous expression of Mtn in Escherichia coli is reported . The metal binding abilities of the Cu- and Zn-MTN complexes conformed in vivo, as well as the features of the Cd- and Cu-aggregates produced by metal replacement in vitro, have been determined by atomic emission spectrometry, circular dichroism and electrospray ionization mass spectrometry . Primary structure relationships with other MT have been examined . The results indicate a close resemblance of MTN to fungal copper-thioneins.

Gene, 2000 Jan 11, 241(2), 213 - 22
Identification and cloning of an aspartyl proteinase from Coccidioides immitis; Johnson SM et al.; A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis . From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases . The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence . The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced . The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E . coli . The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography . This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.

Tohoku J Exp Med, 1999 Nov, 189(3), 191 - 202
Interleukin-13 prevents diaphragm muscle deterioration in a septic animal model; Takahashi Y et al.; The effects of an intravenous injection of Interleukin-13 (IL-13) after endotoxin administration on diaphragm muscle were studied using Wistar rats . Two treatment groups, a control (saline+endotoxin) group and an IL-13 (IL-13+endotoxin) group were studied . E . coli endotoxin (10 mg/kg) was injected intraperitoneally 5 minutes after saline or IL-13 (0.25 microg) injection . The force-frequency curves, twitch kinetics and fatigability were measured at 0 and 4 hours after endotoxin injection . The force-frequency curves and twitch tension in the control group were significantly lower at 4 hours than those at 0 hour due to endotoxin . On the other hand, IL-13 prevented the decrement of the force-frequency curves and twitch tension induced by endotoxin . Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry showed positive staining at 4 hours due to endotoxin in the control group; however, IL-13 also blocked NADPH diaphorase staining at 4 hours . Furthermore, the positive muscle fibers detected by the NADPH diaphorase staining were classified as type I (slow twitch) muscle fibers by ATPase staining . We conclude that IL-13 prevents the deterioration of contraction induced by endotoxin by inhibiting nitric oxide production in the diaphragm muscle, mainly the type I muscle fibers.

J Biotechnol, 2000 Jan 28, 77(1), 65 - 80
Introduction of polyphosphate as a novel phosphate pool in the chloroplast of transgenic potato plants modifies carbohydrate partitioning; van Voorthuysen T et al.; Potato plants (Solanum tuberosum L., cv . Desiree) were transformed with the polyphosphate kinase gene from Escherichia coli fused to the leader sequence of the ferredoxin oxidoreductase gene (FNR) from Spinacea oleracea under the control of the leaf specific St-LS1 promoter to introduce a novel phosphate pool in the chloroplasts of green tissues . Transgenic plants (cpPPK) in tissue culture developed necrotic lesions in older leaves and showed earlier leaf senescence while greenhouse plants showed no noticeable phenotype . Leaves of cpPPK plants contained less starch but higher concentrations of soluble sugars . The presence of polyphosphate in cpPPK leaves was demonstrated by toluidine blue staining and unambiguously verified and quantified by in vitro 31P-NMR of extracts . Polyphosphate accumulated during leaf development from 0.06 in juvenile leaves to 0.83 mg P g-1 DW in old leaves and had an average chain length of 18 residues in mature leaves . In situ 31P-NMR on small leaf pieces perfused with well-oxygenated medium showed only 0.036 mg P g-1 DW polyphosphate that was, however, greatly increased upon treatment with 50 mM ammonium sulfate at pH 7.3 . This phenomenon along with a yield of 0.47 mg P g-1 DW polyphosphate from an extract of the same leaf material suggests that 93% of the polyphosphate pool is immobile . This conclusion is substantiated by the observation that no differences in polyphosphate pool sizes could be discerned between darkened and illuminated leaves, leaves treated with methylviologen or anaerobis and control leaves, treatments causing a change in the pool of ATP available for polyPi synthesis . Results are discussed in the context of the chelating properties of polyphosphates for cations and its consequences for the partitioning of photoassimilate between starch and soluble sugars.

Genes Dev, 2000 Feb 1, 14(3), 360 - 5
RecA protein-dependent R-loop formation in vitro; Kasahara M et al.; The RecA protein of Escherichia coli, which has crucial roles in homologous recombination, DNA damage repair, induction of the SOS response, and SOS mutagenesis, was found to catalyze assimilation of complementary RNA into a homologous region of a DNA duplex (R-loop) . The reaction strictly requires a region of mismatch in the duplex, which may serve as a nucleation site for RecA protein polymerization . The optimum conditions for the assimilation reaction resemble those for the previously studied RecA protein-catalyzed homologous pairing and strand exchange reaction between two DNA molecules . Our finding lends strong support to the proposal that RecA protein-catalyzed assimilation of a transcript into duplex DNA results in formation of an R-loop at certain regions of the chromosome and that, when stabilized, the R-loop can serve as an origin of chromosome replication.

Structure Fold Des, 2000 Feb 15, 8(2), 185 - 95
The 1.8 A crystal structure and active-site architecture of beta-ketoacyl-acyl carrier protein synthase III (FabH) from escherichia coli; Davies C et al.; Background: beta-Ketoacyl-acyl carrier protein synthase III (FabH) initiates elongation in type II fatty acid synthase systems found in bacteria and plants . FabH is a ubiquitous component of the type II system and is positioned ideally in the pathway to control the production of fatty acids . The elucidation of the structure of FabH is important for the understanding of its regulation by feedback inhibition and its interaction with drugs . Although the structures of two related condensing enzymes are known, the roles of the active-site residues have not been experimentally tested . Results: The 1.8 A crystal structure of FabH was determined using a 12-site selenium multiwavelength anomalous dispersion experiment . The active site (Cys112, His244 and Asn274) is formed by the convergence of two alpha helices and is accessed via a narrow hydrophobic tunnel . Hydrogen-bonding networks that include two tightly bound water molecules fix the positions of His244 and Asn274, which are critical for the decarboxylation and condensation reactions . Surprisingly, the His244-->Ala mutation does not affect the transacylation reaction suggesting that His244 has only a minor influence on the nucleophilicity of Cys112 . Conclusions: The histidine and asparagine active-site residues are both required for the decarboxylation step in the condensation reaction . The nucleophilicity of the active-site cysteine is enhanced by the alpha-helix dipole effect, and an oxyanion hole promotes the formation of the tetrahedral transition state.

Structure Fold Des, 2000 Feb 15, 8(2), 123 - 35
Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose; Somoza JR et al.; Background: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose . This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose . Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X . Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII) . Results: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution . GMD is a homodimeric protein with each monomer composed of two domains . The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site . We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose . Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent . GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis . Conclusions: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins . We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function . The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.

Structure Fold Des, 2000 Jan 15, 8(1), 57 - 66
The structure of TolB, an essential component of the tol-dependent translocation system, and its protein-protein interaction with the translocation domain of colicin E9; Carr S et al.; BACKGROUND: E colicin proteins have three functional domains, each of which is implicated in one of the stages of killing Escherichia coli cells: receptor binding, translocation and cytotoxicity . The central (R) domain is responsible for receptor-binding activity whereas the N-terminal (T) domain mediates translocation, the process by which the C-terminal cytotoxic domain is transported from the receptor to the site of its cytotoxicity . The translocation of enzymatic E colicins like colicin E9 is dependent upon TolB but the details of the process are not known . RESULTS: We have demonstrated a protein-protein interaction between the T domain of colicin E9 and TolB, an essential component of the tol-dependent translocation system in E . coli, using the yeast two-hybrid system . The crystal structure of TolB, a procaryotic tryptophan-aspartate (WD) repeat protein, reveals an N-terminal alpha + beta domain based on a five-stranded mixed beta sheet and a C-terminal six-bladed beta-propeller domain . CONCLUSIONS: The results suggest that the TolB-box residues of the T domain of colicin E9 interact with the beta-propeller domain of TolB . The protein-protein interactions of other beta-propeller-containing proteins, the yeast yPrp4 protein and G proteins, are mediated by the loops or outer sheets of the propeller blades . The determination of the three-dimensional structure of the T domain-TolB complex and the isolation of mutations in TolB that abolish the interaction with the T domain will reveal fine details of the protein-protein interaction of TolB and the T domain of E colicins.

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 947 - 52
beta3-endonexin as a novel inhibitor of cyclin A-associated kinase; Ohtoshi A et al.; Cyclin A is indispensable for S phase cell cycle progression and is suggested to be a crucial target of cell adhesion signals . In this study, we demonstrate that beta3-endonexin, a molecule known to associate with the integrin beta3 cytoplasmic domain, specifically binds cyclin A . Deletion of the amino-terminal 52-amino-acid residues including the cyclin-binding RxL motif abolishes the ability of beta3-endonexin to interact with cyclin A . In an in vitro kinase assay, beta3-endonexin inhibits pRB kinase activity associated with cyclin A-Cdk2 while leaving its histone H1 kinase activity unaffected . Coexpression of beta3-endonexin in yeast cells overcomes growth suppression caused by an activation of cyclin A-associated kinase . Our results indicate that beta3-endonexin is a novel cyclin A-binding molecule that regulates cyclin A-associated pRB kinase activity .

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 887 - 91
Cloning and characterization of an alpha-neurotoxin-type protein specific for the coral snake Micrurus corallinus; Silveira de Oliveira J et al.; During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, we cloned an alpha-neurotoxin homologue cDNA (nxh1) . Two others isoforms were also cloned (nxh3 and nxh7, respectively) . The nxh1 cDNA codes for a potential coral snake toxin with a signal peptide of 21 amino acids plus a predicted mature peptide with 57 amino acids . The deduced protein is highly similar to known toxic three-finger alpha-neurotoxins, with four deduced S-S bridges at the same conserved positions . This is the first cDNA coding for a three-finger related protein described so far for coral snakes . However, the predicted protein does not possess some of the important amino acids for the nicotinic acetylcholine receptor interaction . This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein . This protein was used to generate antibodies which recognized the recombinant protein in Western blot and also a single band present in the M . corallinus venom, but not in the venom of 10 other Micrurus species .

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 777 - 82
A novel method for expression and large-scale production of human brain l-glutamate decarboxylase; Davis KM et al.; l-Glutamate decarboxylase (GAD; EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain . Imbalance in the conversion of glutamate to GABA has been implicated in a host of human diseases . Studies on the structure, function, and therapeutic use of GAD have been precluded by insufficient quantities of purified active enzyme . Here we report a novel methodology for the expression and large-scale production of enzymatically active, pure, recombinant human GAD65 and GAD67 . This method circumvents the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins . The availability of sufficient quantities of purified HGAD65 and HGAD67 has allowed for the production of specific polyclonal antibodies that discriminate between the two isoforms . This methodology, in addition to providing key human brain enzymes, may be generally applicable to other systems .

J Hepatol, 2000 Jan, 32(1), 112 - 20
Endotoxin-induced aggravation of preservation-reperfusion injury of rat liver and its modulation; Vajdova K et al.; BACKGROUND/AIMS: In clinical transplantation, exposure of donors to gut-derived endotoxin occurs frequently and may adversely affect liver transplantation therapy . The aim of this study was to investigate: 1) whether brief exposure of rats to endotoxin before liver procurement aggravates the early phase of reperfusion injury of hepatic explants; and if so 2) whether Kupffer cell activation is a contributing factor to liver injury; and 3) whether heparin and pentoxifylline could minimize this effect . METHODS: Male Wistar rats were injected with 0.2-4.0 mg/kg of Escherichia coli lipopolysaccharide 2 h prior to liver harvest . After preservation in University of Wisconsin cold-storage solution, the livers were reperfused using a blood-free perfusion model . To inactivate Kupffer cells, some rats were pretreated with gadolinium chloride or liposome-encapsulated dichloromethylene-diphosphonate before lipopolysaccharide administration . The other rats received lipopolysaccharide with heparin or pentoxifylline . RESULTS: In a dose-independent fashion, lipopolysaccharide impaired portal flow during graft reperfusion . In a dose-dependent way, lipopolysaccharide increased lactate dehydrogenase release into the perfusate and decreased bile flow and bromosulfophthalein excretion . Gadolinium chloride, liposomal dichloromethylene-diphosphonate, heparin, and pentoxifylline reduced lactate dehydrogenase release by 34%, 43%, 59%, and 64%, respectively, and improved functional parameters of the liver . A 52-fold increased neutrophil infiltration in the liver sinusoids after lipopolysaccharide exposure was not affected significantly by the drugs studied; however, heparin reduced markedly neutrophil activation . CONCLUSIONS: The results of this investigation provide direct evidence that aggravation of preservation-reperfusion injury of rat liver by endotoxin is mediated by Kupffer cell-dependent mechanism(s) and it can be minimized by heparin and pentoxifylline.

J Hepatol, 2000 Jan, 32(1), 25 - 31
Enhanced monocyte activation and hepatotoxicity in response to endotoxin in portal hypertension; Perez del Pulgar S et al.; BACKGROUND/AIMS: Septic shock is a systemic response to infection, and it causes a high mortality rate in cirrhotic patients . The mechanisms responsible for this susceptibility in cirrhosis are poorly understood . The aim of this study was to investigate whether monocyte activation and hepatic function are altered in portal hypertension after endotoxin administration . METHODS: Portal-hypertensive and sham-operated rats were used . Plasma levels of tumor necrosis factor-alpha after lipopolysaccharide stimulation (both in vivo and in vitro) were measured by ELISA . CD11b/CD18 integrin expression on leukocyte membrane was measured by flow cytometry . Plasma transaminase activities were also determined . RESULTS: The levels of tumor necrosis factor-alpha in plasma and the expression of CD11b/CD18 on leukocytes in portal-hypertensive rats was similar to that in sham-operated rats . Injection of 150 microg/kg of lipopolysaccharide produced a 9-fold increase in plasma levels of tumor necrosis factor-alpha in portal-hypertensive compared with sham-operated rats, together with a significant up-regulation of CD11b/CD18 expression on monocytes and an elevation in plasma transaminase activity . Blood leukocytes incubated in vitro with lipopolysaccharide (0.5 microg/ml) induced a hypersecretion of tumor necrosis factor-alpha in portal-hypertensive rats, as compared to sham-operated rats . CONCLUSIONS: This study shows that monocytes from portal-hypertensive rats have an enhanced response to endotoxin, leading to hepatotoxicity.

Biochim Biophys Acta, 1999 Dec 23, 1489(2-3), 457 - 61
Genetic analysis of the gene cluster responsible for synthesis of serotype e-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans; Yoshida Y et al.; A gene cluster associated with the biosynthesis of the serotype e-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans IDH1705 belonging to serotype e was cloned and sequenced . This cluster consisted of 18 open reading frames . Escherichia coli produced the polysaccharide that reacts with the serotype e-specific antiserum when transformed with a plasmid containing the cluster . Comparing the structure of the gene cluster with similar clusters from A . actinomycetemcomitans strains Y4 (serotype b) and NCTC9710 (serotype c) revealed that a 5.3-kb region containing the distal half of one gene and two entire genes in the cluster from strain IDH1705 replaced a 6.2-kb region containing eight genes in the cluster from strain Y4, and a 4.7-kb region containing four genes in the cluster from strain NCTC9710 . These results suggest that this region is essential to the antigenic specificity of serotype e A . actinomycetemcomitans.

Res Microbiol, 1999 Nov-Dec, 150(9-10), 653 - 64
Novel intergenic repeats of Escherichia coli K-12; Rudd KE; An online catalog of intergenic DNA repeat sequence elements is added to the EcoGene Escherichia coli K-12 genome sequence annotation and analysis project (bmb.med.miami.edu/EcoGene) . A library of noncoding (intergenic) DNA sequences depleted of known intergenic repeat classes was searched for DNA sequence similarities to identify novel DNA repeat sequence classes.

Mol Cells, 1999 Dec 31, 9(6), 587 - 95
Generation of self-antigen reactive, anti-urocortin specific antibodies by immunization of recombinantly expressed urocortin fusion proteins; Na SY et al.; Urocortin is a recently described 40-meric neuropeptide, which was originally detected in the rat mid-brain and is believed to play a key role in response to stress situations . While its function in the central nervous system is rather well established, the biological role in the periphery is still to be determined . To investigate its distribution and effect on peripheral cells and tissues, in the present study, urocortin was recombinantly expressed and specific antibodies were generated . So far, the immunological detection of urocortin in the rat was largely dependent on antisera generated in rabbits . However, the polyclonal nature of the serum and the remote species origin tend to show cross-reactivities and higher backgrounds . On the other hand, generation of mouse antibodies to rat urocortin was hampered since mouse and rat urocortin sequences are identical, and such antibodies would represent auto-reactive antibodies . Despite such restrictions, the immunization with a combination of various recombinantly expressed urocortin fusion proteins resulted in the successful generation of mouse antiurocortin antisera, whose specificities were confirmed by ELISA and Western blot analysis . To produce the recombinant proteins for immunization, a cDNA encoding the mature urocortin sequence was cloned and expressed in fusion either with the glutathione-S-transferase, the maltose-binding protein, thioredoxin, or a 6X His tag . Depending on the expression system, the solubility and yield of the recombinant proteins greatly varied . Together with the newly generated antibodies, these recombinantly expressed urocortin proteins will serve as valuable tools in further investigations of the biological function of urocortin.

Acta Virol, 1999 Feb, 43(1), 31 - 7
Accumulation of helper component/proteinase and coat protein of turnip mosaic virus in intact plants; Ohshima K; The helper component/proteinase (HC/Pro) protein of turnip mosaic virus (TuMV) was fused with glutathione S-transferase (GST) and expressed as a fusion protein in Escherichia coli . The quality of antiserum raised against the GST-HC/Pro fusion protein was compared to that of antiserum raised against coat protein (CP) by image analyser . The result showed that these antisera were of similar quality . Then the both antisera were used to follow the time course of accumulation of HC/Pro protein and CP in intact TuMV-infected leaves . CP appeared first at day 3 post inoculation (p.i.) and gradually accumulated in uninoculated upper leaves, whereas HC/Pro protein appeared first at day 4 p.i., accumulated up to day 7 p.i . and then gradually decreased . Potyvirus proteins are encoded by a single translation unit spanning most of the genome and are presumably synthesized in equimolar ratios . Therefore, the reduced accumulation of HC/Pro protein in relation to CP at one month p.i . in infected plants is presumed to be the result of its degradation.

Mol Microbiol, 2000 Feb, 35(3), 657 - 66
The response regulator RssB, a recognition factor for sigmaS proteolysis in Escherichia coli, can act like an anti-sigmaS factor; Becker G et al.; sigmaS (RpoS) is the master regulator of the general stress response in Escherichia coli . Several stresses increase cellular sigmaS levels by inhibiting proteolysis of sigmaS, which under non-stress conditions is a highly unstable protein . For this ClpXP-dependent degradation, the response regulator RssB acts as a recognition factor, with RssB affinity for sigmaS being modulated by phosphorylation . Here, we demonstrate that RssB can also act like an anti-sigma factor for sigmaS in vivo, i.e . RssB can inhibit the expression of sigmaS-dependent genes in the presence of high sigmaS levels . This becomes apparent when (i) the cellular RssB/sigmaS ratio is at least somewhat elevated and (ii) proteolysis is reduced (for example in stationary phase) or eliminated (for example in a clpP mutant) . Two modes of inhibition of sigmaS by RssB can be distinguished . The 'catalytic' mode is observed in stationary phase cells with a substoichiometric RssB/sigmaS ratio, requires ClpP and therefore probably corresponds to sequestering of sigmaS to Clp protease (even though sigmaS is not degraded) . The 'stoichiometric' mode occurs in clpP mutant cells upon overproduction of RssB to levels that are equal to those of sigmaS, and therefore probably involves binary complex formation between RssB and sigmaS . We also show that, under standard laboratory conditions, the cellular level of RssB is more than 20-fold lower than that of sigmaS and is not significantly controlled by stresses that upregulate sigmaS . We therefore propose that antisigma factor activity of RssB may play a role under not yet identified growth conditions (which may result in RssB induction), or that RssB is a former antisigma factor that during evolution was recruited to serve as a recognition factor for proteolysis.

Mol Microbiol, 2000 Feb, 35(3), 577 - 88
The Brucella abortus Lon functions as a generalized stress response protease and is required for wild-type virulence in BALB/c mice; Robertson GT et al.; The gene encoding a Lon protease homologue has been cloned from Brucella abortus . The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB:lacZ fusion to wild-type levels . A sigma32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses . ATP-dependent proteolytic activity was also demonstrated for purified recombinant Lon . To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange . In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41 degrees C and displayed an increased sensitivity to killing by puromycin and H2O2 . GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain . Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice . These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild-type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.

Insect Mol Biol, 2000 Feb, 9(1), 47 - 55
Cloning and characterization of the ribosomal protein CcP0 of the medfly Ceratitis capitata; Gagou M et al.; The gene of the ribosomal protein CcP0, the third member of the ribosomal P-protein family of the medfly Ceratitis capitata, was identified by genomic and cDNA sequence analysis . It codes for a polypeptide of 317 amino acids and its predicted amino acid sequence shows great similarity to the P0 proteins of other eukaryotic organisms . The CcP0 gene was expressed in Escherichia coli and the 34-kDa recombinant protein was identical to the P0 protein of purified medfly ribosomes . Both proteins reacted positively with a specific monoclonal antibody against the highly conserved C terminus of eukaryotic ribosomal P proteins . Interestingly, the medfly CcP0 seems to be the only P0 protein of higher eukaryotic organisms with basic character (pI 8.5), as shown by electrofocusing of purified ribosomes.

Genes Cells, 2000 Feb, 5(2), 101 - 9
Control of genetic stability in Escherichia coli: the SbcB 3'-5' exonuclease suppresses illegitimate recombination promoted by the RecE 5'-3' exonuclease; Yamaguchi H et al.; BACKGROUND: The Escherichia coli sbcB gene, which codes for a 3'-5' exonuclease, ExoI, is known to suppress illegitimate recombination . In contrast, the recE gene, which codes for a 5'-3' exonuclease, Exo VIII promotes joining between DNA ends having short stretches of homology . Therefore, it seems likely that the 3'-5' and 5'-3' exonucleases regulate genetic instability that is mediated by illegitimate recombination . However, there has been little evidence to substantiate the involvement of exonuclease activity in the promotion and suppression of illegitimate recombination . RESULTS: Using a plasmid system for the analysis of deletion formation, we first demonstrated that deletion formation is increased by the sbcA mutation, which activates the expression of RecE 5'-3' exonuclease . It is thought that DNA ends having 3'-single stranded overhangs are important for illegitimate recombination . Next, we found that a large supply of SbcB 3'-5' exonuclease suppresses the deletion formation enhanced by the RecE exonuclease . Moreover, the SbcB exonuclease even suppressed deletion formation in cells not expressing RecE exonuclease . CONCLUSION: We conclude that DNA ends with 3'-overhangs produced by 5'-3' dsDNA exonuclease activity are proficient for illegitimate recombination, while blunt DNA ends produced by 3'-5' ssDNA exonuclease activity are deficient for illegitimate recombination . Therefore, both exonucleases may play important roles in genetic stability by controlling end-joining between DNA molecules.

Eur J Biochem, 2000 Feb, 267(4), 1125 - 37
The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 degrees C; Spies M et al.; The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair . We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity . The purified P . islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C . It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH . P . islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange . At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA . The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C . The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature . These results suggest that the ssDNA-dependent ATPase activity of P . islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature.

Eur J Biochem, 2000 Feb, 267(4), 1068 - 74
Cytochrome cM from synechocystis 6803 . Detection in cells, expression in Escherichia coli, purification and physical characterization; Cho YS et al.; Based on DNA sequence data a novel c-type cytochrome, cytochrome cM, has been predicted to exist in the cyanobacterium Synechocystis 6803 . The precursor protein consists of 105 amino acids with a characteristic heme-binding motif and a hydrophobic domain located at the N-terminal end that is proposed to act as either a signal peptide or a membrane anchor . For the first time we report the detection of cytochrome cM in Synechocystis 6803 using Western blot analysis . The soluble portion cytochrome cM has been overexpressed in Escherichia coli in two forms, one with a poly histidine tag to facilitate purification and one without such a tag . The overexpressed protein has been purified and shown to bind heme, exhibiting an absorption peak in the Soret band near 416 nm and a peak in the alpha band at 550 nm . The extinction coefficient of cytochrome cM is 23.2 +/- 0.5 mM-1.cm-1 for the reduced minus oxidized alpha band peak (550-535 nm) . The isoelectric point of cytochrome cM is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.2 predicted from the amino acid sequence . The redox midpoint potential of cytochrome cM expressed in E . coli is 151 +/- 5 mV (pH 7.1), which is quite low compared to other c-type cytochromes in which a histidine and a methionine residue serve as the axial ligands to the heme . This work opens the way for determining the three-dimensional structure of cytochrome cM and investigating its function in cyanobacteria.

J Biol Chem, 2000 Feb 18, 275(7), 5120 - 3
Catalytic sites for 3' and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC; Verhoeven EE et al.; Nucleotide excision repair in Escherichia coli is a multistep process in which DNA damage is removed by incision of the DNA on both sides of the damage, followed by removal of the oligonucleotide containing the lesion . The two incision reactions take place in a complex of damaged DNA with UvrB and UvrC . It has been shown (Lin, J . -J., and Sancar, A . (1992) J . Biol . Chem . 267, 17688-17692) that the catalytic site for incision on the 5' side of the damage is located in the UvrC protein . Here we show that the catalytic site for incision on the 3' side is in this protein as well, because substitution R42A abolishes 3' incision, whereas formation of the UvrBC-DNA complex and the 5' incision reaction are unaffected . Arg(42) is part of a region that is homologous to the catalytic domain of the homing endonuclease I-TevI . We propose that the UvrC protein consists of two functional parts, with the N-terminal half for the 3' incision reaction and the C-terminal half containing all the determinants for the 5' incision reaction.

J Biol Chem, 2000 Feb 18, 275(7), 5104 - 10
Cloning and expression of glycolipid transfer protein from bovine and porcine brain; Lin X et al.; Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids . Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs . The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR . The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR . The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation . The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine . Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein . Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active . Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP . Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle . Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.

J Biol Chem, 2000 Feb 18, 275(7), 5073 - 80
The differentially conserved residues of carbamoyl-phosphate synthetase; Javid-Majd F et al.; Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimeric protein . The larger of the two subunits (M(r) approximately 118,000) contains a pair of homologous domains of approximately 400 residues each that are approximately 40% identical in amino acid sequence . The carboxy phosphate (residues 1-400) and carbamoyl phosphate domains (residues 553-933) also contain approximately 79 differentially conserved residues . These are residues that are conserved throughout the bacterial evolution of CPS in one of these homologous domains but not the other . The role of these differentially conserved residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other . Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized . A majority of the mutants were unable to synthesize any carbamoyl phosphate and the rest were severely crippled . True tandem repeat chimeric proteins were constructed by the complete substitution of one homologous domain sequence for the other . Neither of the two possible chimeric proteins was structurally stable . These results have been interpreted to demonstrate that the two homologous domains in the large subunit of CPS are functionally and structurally nonequivalent . This nonequivalence is a direct result of the specific functions each of these domains must perform during the overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved residues.

J Biol Chem, 2000 Feb 18, 275(7), 4956 - 64
Distinct repair activities of human 7,8-dihydro-8-oxoguanine DNA glycosylase and formamidopyrimidine DNA glycosylase for formamidopyrimidine and 7,8-dihydro-8-oxoguanine; Asagoshi K et al.; 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells . Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively . In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site . The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg . However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg . Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference . hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases . Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair . The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg . The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.

J Biol Chem, 2000 Feb 18, 275(7), 4906 - 11
A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis; Cheng YQ et al.; The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungus Cochliobolus carbonum and an inhibitor of histone deacetylase . The major form of HC-toxin contains the D-isomers of Ala and Pro . The non-ribosomal peptide synthetase that synthesizes HC-toxin has only one epimerizing domain for conversion of L-Pro to D-Pro; the source of D-Ala has remained unknown . Here we present the cloning and characterization of a new gene involved in HC-toxin biosynthesis, TOXG . TOXG is present only in HC-toxin-producing (Tox2(+)) isolates of C . carbonum . TOXG is able to support D-Ala-independent growth of a strain of Escherichia coli defective in D-Ala synthesis . A C . carbonum strain with both of its copies of TOXG mutated grows normally in culture, and although it no longer makes the three forms of HC-toxin that contain D-Ala, it still makes a minor form of HC-toxin that contains Gly in place of D-Ala . The addition of D-Ala to the culture medium restores production of the D-Ala-containing forms of HC-toxin by the toxG mutant . The toxG mutant has only partially reduced virulence . It is concluded that TOXG encodes an alanine racemase whose function is to synthesize D-Ala for incorporation into HC-toxin.

J Biol Chem, 2000 Feb 18, 275(7), 4734 - 42
The monoclonal antibody 1F6 identifies a pH-dependent conformational change in the hydrophilic NH(2) terminus of NhaA Na(+)/H(+) antiporter of Escherichia coli; Venturi M et al.; One of the most interesting properties of the NhaA Na(+)/H(+) antiporter of Escherichia coli is the strong regulation of its activity by pH . This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX . In the present work we show that a monoclonal antibody (mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner . This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAbs bind to NhaA independently of pH . The epitope of mAb 1F6 was located at the NH(2) terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing . The antibody bound to the peptide HLHRFFSS, starting at the third amino acid of NhaA . A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesting that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH . Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na(+)/H(+) antiporter activity as well as to its sensitivity to trypsin . We thus suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter . Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA . Furthermore, the binding site of mAb 1F6 is involved in the functioning of the antiporter as follows: a double Cys replacement H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

J Bacteriol, 2000 Mar, 182(5), 1427 - 31
Requirement for homologous recombination functions for expression of the mutA mistranslator tRNA-induced mutator phenotype in Escherichia coli; Ren L et al.; Expression of the Escherichia coli mutA mutator phenotype requires recA, recB, recC, ruvA, and ruvC gene, but not recD, recF, recO, or recR genes . Thus, the recBCD-dependent homologous recombination system is a component of the signal pathway that activates an error-prone DNA polymerase in mutA cells.

J Bacteriol, 2000 Mar, 182(5), 1423 - 6
Cpx two-component signal transduction in Escherichia coli: excessive CpxR-P levels underlie CpxA* phenotypes; De Wulf P et al.; In Escherichia coli, the CpxA-CpxR two-component signal transduction system and the sigma(E) and sigma(32) response pathways jointly regulate gene expression in adaptation to adverse conditions . These include envelope protein distress, heat shock, oxidative stress, high pH, and entry into stationary phase . Certain mutant versions of the CpxA sensor protein (CpxA* proteins) exhibit an elevated ratio of kinase to phosphatase activity on CpxR, the cognate response regulator . As a result, CpxA* strains display numerous phenotypes, many of which cannot be easily related to currently known functions of the CpxA-CpxR pathway . It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of CpxR . We here report that all of the tested CpxA* phenotypes depend on elevated levels of CpxR-P and not on cross-signalling of CpxA* to noncognate response regulators.

J Bacteriol, 2000 Mar, 182(5), 1419 - 22
Effects of calcium and calcium chelators on growth and morphology of Escherichia coli L-form NC-7; Onoda T et al.; Growth of a wall-less, L-form of Escherichia coli specifically requires calcium, and in its absence, cells ceased dividing, became spherical, swelled, developed large vacuoles, and eventually lysed . The key cell division protein, FtsZ, was present in the L-form at a concentration five times less than that in the parental strain . One interpretation of these results is that the L-form possesses an enzoskeleton partly regulated by calcium.

J Bacteriol, 2000 Mar, 182(5), 1280 - 5
Autophosphorylation of phosphoglucosamine mutase from Escherichia coli; Jolly L et al.; Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, an essential step in the pathway for UDP-N-acetylglucosamine biosynthesis in bacteria . This enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving glucosamine-1, 6-diphosphate as an intermediate (L . Jolly, P . Ferrari, D . Blanot, J . van Heijenoort, F . Fassy, and D . Mengin-Lecreulx, Eur . J . Biochem . 262:202-210, 1999) . However, the process by which the initial phosphorylation of the enzyme is achieved in vivo remains unknown . Here we show that the phosphoglucosamine mutase from Escherichia coli autophosphorylates in vitro in the presence of {(32)P}ATP . The same is observed with phosphoglucosamine mutases from other bacterial species, yeast N-acetylglucosamine-phosphate mutase, and rabbit muscle phosphoglucomutase . Labeling of the E . coli GlmM enzyme with {(32)P}ATP requires the presence of a divalent cation, and the label is subsequently lost when the enzyme is incubated with either of its substrates . Analysis of enzyme phosphorylation by high-pressure liquid chromatography and coupled mass spectrometry confirms that only one phosphate has been covalently linked to the enzyme . Only phosphoserine could be detected after acid hydrolysis of the labeled protein, and site-directed mutagenesis of serine residues located in or near the active site identifies the serine residue at position 102 as the site of autophosphorylation of E . coli GlmM.

J Bacteriol, 2000 Mar, 182(5), 1232 - 42
Characterization and role of tbuX in utilization of toluene by Ralstonia pickettii PKO1; Kahng HY et al.; The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons . The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon . It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds . In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon . The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX . The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli . Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R . pickettii PKO1 . In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase . This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.

Methods, 2000 Feb, 20(2), 219 - 31
Identification of connexin-interacting proteins: application of the yeast two-hybrid screen; Jin C et al.; Protein-protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs . The yeast two-hybrid screen has become a valuable tool for identifying protein-protein interactions . The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control . To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins . We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as "bait." We also describe additional methods to confirm the interactions between Cx43 and the identified proteins . These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy .

Shock, 2000 Feb, 13(2), 117 - 25
Pulmonary endothelial and epithelial integrity and neutrophil infiltration after endotoxin in interleukin-1 receptor knockout mice; Sutton ET et al.; Previously we found the structural integrity of the aortic endothelium was maintained after the administration of endotoxin in type 1 interleukin-1 (IL-1) receptor knockout mice . In this study, we investigated further the integrity of pulmonary vascular endothelium, airway epithelial, pulmonary microvasculature, and neutrophil infiltration into the microvasculature and respiratory air spaces . Adult male C57BL/129J wild-type mice and C57BL/129J knockout mice possessing a homozygous deletion of the type 1 IL-1 receptor received the following intraperitoneal injections; 1) Escherichia coli endotoxin (ENDT) (10 mg/kg), 2) ENDT (2 mg/kg given for 4 days), or (3) saline vehicle . Wild-type and knockout control animals receiving saline vehicle showed normal endothelial and epithelial ultrastructure with intact membranes . Pulmonary endothelial cell damage was found only in wild-type mice given a single 10 mg/kg endotoxin dose . Airway epithelial damage was found only in wild-type mice given a repetitive dose of endotoxin (2 mg/kg for 4 days) . Neutrophil infiltration increased only in mice given a single dose of endotoxin (10 mg/kg) with the wild-type increasing by 32% and the knockouts by 6% compared with the saline control for that group respectively . Serum IL-6 and nitric oxide (indicators of septic shock severity and lethality) significantly increased only in the mice given 10 mg/kg of endotoxin . The maintenance of pulmonary endothelial and epithelial cell integrity and the decrease of neutrophil infiltration in the IL-1 knockout mice suggest that IL-1 contributes significantly to the severity of endotoxin-induced sepsis.

J Med Microbiol, 2000 Feb, 49(2), 149 - 55
Development and evaluation of a solid-phase enzyme immunoassay based on Andes hantavirus recombinant nucleoprotein; Padula PJ et al.; Hantavirus pulmonary syndrome (HPS) with high mortality rate has been reported in five countries in South America . Rapid accurate methods are important both for monitoring acute infections and for epidemiological studies . The Andes virus nucleoprotein amino acid sequence has a high identity percentage compared with other sequences of this region and has been chosen for the development of diagnostic reagents . Andes nucleoprotein expressed in Escherichia coli was applied as antigen in IgG, IgA and mu-capture IgM enzyme-linked inmunosorbent assays (ELISAs) . An evaluation of this reagent was conducted to establish its usefulness for differential diagnosis of HPS and seroprevalence studies . Samples from 135 reverse transcription (RT)-PCR-confirmed HPS cases, 77 individuals with other respiratory infections and 957 healthy inhabitants from endemic and non-endemic areas were analysed . The hantavirus-infected patients had an early and strong IgM, IgG and IgA serum antibody response, in most of the cases as early as 1, 7 and 1 days following onset of symptoms, respectively . IgM and IgG detection showed a specificity and sensitivity of 100% . Andes-specific IgM antibodies were found in all patients in the first available sample, which remained detectable for at least 43 days . Specific IgA antibodies were also detected in saliva of patients with acute HPS . The short duration of the disease and the risk for contacts due to person-to-person transmission of Andes virus necessitate the use of highly sensitive tests which might lead to earlier detection of infected people and improve the treatment and management of patients with HPS.

Philos Trans R Soc Lond B Biol Sci, 1999 Dec 29, 354(1392), 1977 - 84
Structural biology; Holmes KC; Protein crystallography has become a major technique for understanding cellular processes . This has come about through great advances in the technology of data collection and interpretation, particularly the use of synchrotron radiation . The ability to express eukaryotic genes in Escherichia coli is also important . Analysis of known structures shows that all proteins are built from about 1000 primeval folds . The collection of all primeval folds provides a basis for predicting structure from sequence . At present about 450 are known . Of the presently sequenced genomes only a fraction can be related to known proteins on the basis of sequence alone . Attempts are being made to determine all (or as many as possible) of the structures from some bacterial genomes in the expectation that structure will point to function more reliably than does sequence . Membrane proteins present a special problem . The next 20 years may see the experimental determination of another 40,000 protein structures . This will make considerable demands on synchrotron sources and will require many more biochemists than are currently available . The availability of massive structure databases will alter the way biochemistry is done.

Biochim Biophys Acta, 2000 Feb 9, 1476(2), 324 - 30
Probing the interaction between N(1),N(4)-dibenzylputrescine and tRNA through (15)N NMR: biological implications; Fernandez CO et al.; NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA . The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured . From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined . The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction . This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.

Biochim Biophys Acta, 2000 Feb 9, 1476(2), 311 - 23
Primary structure determinants of the pH- and temperature-dependent aggregation of thioredoxin; Lemaire SD et al.; Thioredoxins are small proteins found in all living organisms . We have previously reported that Chlamydomonas reinhardtii thioredoxin h exhibited differences both in its absorption spectrum and its aggregation properties compared to thioredoxin m . In this paper, we demonstrate, by site-directed mutagenesis, that the particularity of the absorption spectrum is linked to the presence of an additional tryptophan residue in the h isoform . The pH and temperature dependence of the aggregation of both thioredoxins has been investigated . Our results indicate that the aggregation of TRX is highly dependent on pH and that the differences between the two TRX isoforms are linked to distinct pH dependencies . We have also analyzed the pH and temperature dependence of 12 distinct variants of TRX engineered by site-directed mutagenesis . The results obtained indicate that the differences in the hydrophobic core of the two TRX isoforms do not account for the differences of aggregation . On the other hand, we show the importance of His-109 as well as the second active site cysteine, Cys-39 in the aggregation mechanism.

Biochim Biophys Acta, 2000 Feb 9, 1476(2), 239 - 52
Escherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn(2+) holoenzyme are similar; Porter DJ; Recombinant Escherichia coli cytosine deaminase is purified as a mixture of Zn(2+) and Fe(2+) forms of the enzyme . Fe(2+) is removed readily by o-phenanthroline to yield apoenzyme (apoCDase) that contains <0.2 mol of Zn(2+)per mol of subunit . ApoCDase was efficiently reconstituted to Zn(2+)CDase by treatment with ZnCl(2) . The interaction of cytosine with apoCDase and Zn(2+)CDase was investigated at pH 7.5 and 25 degrees C by monitoring changes in intrinsic protein fluorescence . The values for the kinetic data K(1), k(2), and k(3) for Zn(2+)CDase were 0.25 mM, 80 s(-1), and 38 s(-1), respectively . The value for k(-2) was statistically indistinguishable from zero . The analogous values for K(1), k(2), and k(-2), (k(3)=0) for apoCDase were 0.157 mM, 186 s(-1) and approximately 0.8 s(-1), respectively . The overall dissociation constant of apoCDase for cytosine was 0.00069 mM, whereas the K(m) of Zn(2+)CDase for cytosine was 0.20 mM . The pre-steady state phase of the reaction was associated with an absorbance increase at 280 nm that was attributed to solvent perturbation of the spectrum of cytosine or enzyme . Formation of the Fe(2+)CDase-cytosine complex was too rapid to monitor by these techniques.

Biochim Biophys Acta, 2000 Feb 9, 1476(2), 191 - 202
Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes; Bera-Maillet C et al.; The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed . EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene . The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E . coli as well as its detection in F . succinogenes cultures . Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies . One of the polyclonal antipeptide antisera was used to purify EGB . EGC was purified by affinity chromatography with Ni-NTA resin . The endo mode of action of the two enzymes on carboxymethyl-cellulose was different . The values of K(m) and V(max) were respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC . The reactivity of the antipeptide and the anti-EGC sera with F . succinogenes proteins of molecular mass different from that of EGB and EGC produced in E . coli suggested post-translational modification of the two enzymes in F . succinogenes cultures . Expression of endB and endC genes in F . succinogenes was confirmed by RT-PCR.

J Mol Biol, 2000 Feb 18, 296(2), 673 - 84
Dimerisation mutants of Lac repressor . II . A single amino acid substitution, D278L, changes the specificity of dimerisation; Spott S et al.; Assembly of the lactose repressor tetramer involves two subunit interfaces, the C-terminal heptad repeats, and the monomer-monomer interface . Dimerisation between two monomers of Lac repressor of Escherichia coli lacking the two C-terminal heptad repeats occurs through the interactions between three alpha-helices of each monomer, which form a highly hydrophobic interface . Residues possibly involved in specific dimer formation are known from X-ray studies and from the phenotypes of more than 4000 single amino acid substitutions . During the examination of numerous mutants within the dimerisation interface of Lac repressor, we found that substitution of one amino acid, D278 to leucine, is sufficient to change the specificity of dimerisation . Analysis of this single substitution indicates that D278L mutant Lac repressor represses like wild-type . However, it no longer forms heterodimers with wild-type Lac repressor .

J Mol Biol, 2000 Feb 18, 296(2), 569 - 77
Structures of adenylosuccinate synthetase from Triticum aestivum and Arabidopsis thaliana; Prade L et al.; Catalyzing the first step in the de novo synthesis of adenylmonophosphate, adenylosuccinate synthetase (AdSS) is a known target for herbicides and antibiotics . We have purified and crystallized recombinant AdSS from Arabidopsis thaliana and Tritium aestivum, expressed in Escherichia coli . The structures of A . thaliana and T . aestivum AdSS in complex with GDP were solved at 2.9 A and 3.0 A resolution, respectively . Comparison with the known structures from E . coli reveals that the overall fold is very similar to that of the E . coli protein . The longer N terminus in the plant sequences is at the same place as the longer C terminus of the E . coli sequence in the 3D structure . The GDP-binding sites have one additional hydrogen-bonding partner, which is a plausible explanation for the lower K(m) value . Due to its special position, this partner may also enable GTP to initiate a conformational change, which was, in E . coli AdSS, exclusively activated by ligands at the IMP-binding site . The dimer interfaces show up to six hydrogen bonds and six salt-bridges more than in the E . coli structure, although the contact areas have approximately the same size .

J Mol Biol, 2000 Feb 18, 296(2), 449 - 57
Direct visualisation of conformational changes in EF(0)F(1) by electron microscopy; Bottcher B et al.; The isolated H(+)-ATPase from Escherichia coli (EF(0)F(1)) was investigated by electron microscopy of samples of negatively stained monodisperse molecules, followed by single-particle image processing . The resulting three-dimensional maps showed that the F(1)-part is connected by a prominent stalk to a more peripheral part of F(0) . The F(1)-part showed stain-accessible cavities inside . In three-dimensional maps from selected particles, a second stalk could be detected which was thinner than the main stalk and is thought to correspond to the stator.Three-dimensional maps of the enzyme in the absence and in the presence of the substrate analogue adenyl-beta, gamma-imidodiphosphate (AMP-PNP) were calculated . Upon binding of AMP-PNP the three-dimensional maps showed no significant changes in the F(0)-part of EF(0)F(1), whereas a major conformational change in the F(1)-part was observed . (1) The diameter of the F(1)-part decreased upon binding of AMP-PNP mainly in the upper half of F(1) . (2) Enzyme particles prepared in the presence of AMP-PNP had a pointed cap at the top of the F(1)-part which was missing in its absence . (3) The stain-accessible cavity inside the F(1)-part altered its pattern significantly .

J Mol Biol, 2000 Feb 18, 296(2), 403 - 19
Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase; Lee J et al.; Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time . The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate . The conformational dynamics by which the recombination complex "isomerizes" from the Holliday-forming to the Holliday-resolving mode are not well understood . Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction . We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering . We have not observed a strong resolution bias in favor of the strands designed to assume the "crossed" configuration within the unbound junction . The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity . On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends .

J Allergy Clin Immunol, 2000 Feb, 105(2 Pt 1), 279 - 85
Escherichia coli expression and purification of recombinant dog albumin, a cross-reactive animal allergen; Pandjaitan B et al.; BACKGROUND: Animal hair-dander represents an important source of indoor allergens . Diagnosis and therapy of animal allergy would benefit from the availability of defined recombinant allergens . They may be preferred to animal-derived proteins, particularly for in vivo application in patients . OBJECTIVE: The purpose of this study was to express and purify recombinant dog albumin, an important cross-reactive animal allergen . METHODS: Complementary (c)DNA sequences coding for dog albumin were obtained by reverse transcription and subsequent PCR amplification from dog liver RNA . Dog albumin-encoding cDNA sequences were inserted into phage lambdagt11, and IgE-reactive phage clones were isolated by immunoscreening with serum IgE from a patient with dog allergy . Dog albumin was expressed as IgE-reactive recombinant protein in Escherichia coli and purified by inclusion body preparation, resolubilization, and diethylaminoethyl cellulose chromatography . Cross-reactivity of dog albumin with cat and human albumin was examined by immunoblot, as well as ELISA inhibition experiments . RESULTS: A cDNA sequence coding for complete dog albumin was obtained by reverse transcription and subsequent PCR amplification from dog liver . The cDNA and deduced amino acid sequence of dog albumin was highly homologous to the sequences of albumins from animals to human subjects, thus explaining the extensive cross-reactivities among albumins . Recombinant dog albumin was expressed in E coli and purified . It reacted with serum IgE from patients allergic to dog albumin and a monoclonal anti-human albumin antibody . Immunologic competition experiments performed with serum IgE from patients allergic to dog albumin and a mouse monoclonal antihuman albumin antibody indicated the presence of similar epitopes on dog, cat, and human albumin . CONCLUSION: Recombinant dog albumin may be used for diagnostic purposes to identify patients who are cross-sensitized to many animal species and perhaps for specific immunotherapy of sensitized individuals.

Biotechnol Appl Biochem, 2000 Feb, 31 ( Pt 1), 21 - 7
Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin; Guo L et al.; Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library . The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment . The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E . coli BL21 (DE3) as inclusion bodies . The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE . It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C . The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.

J Infect Dis, 2000 Feb, 181(2), 774 - 8
Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coli; Langermann S et al.; Escherichia coli FimH adhesin mediates binding to the bladder mucosa . In mice, a FimH vaccine protects against bacterial challenge . In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly . After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys . All 8 monkeys were challenged with 1 mL of 108 E . coli cystitis isolate NU14 . Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected . These findings suggest that a vaccine based on the FimH adhesin of E . coli type 1 pili may have utility in preventing cystitis in humans.

J Chromatogr A, 1999 Dec 24, 864(2), 247 - 56
Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose; Chaga G et al.; A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins . The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells . Each protein was expressed, extracted and purified in one chromatographic step . The purification procedure for each protein can be accomplished in less than 1 h . A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min . The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis . Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity . The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined.

Acta Anaesthesiol Scand, 2000 Jan, 44(1), 24 - 31
Effects of melagatran, an inhibitor of thrombin, on fibrin deposits, haemodynamics, and platelet count in endotoxaemic pigs; Eriksson M et al.; BACKGROUND: Thrombin plays a pivotal role in the development of septic shock . Porcine endotoxaemia can replicate this condition . We wanted to evaluate whether melagatran, a novel inhibitor of thrombin, would counteract some of the endotoxin-induced changes in this model . METHODS: Fifteen pigs were anaesthetised, monitored (circulatory and respiratory variables) and subjected to an infusion of E . coli endotoxin at 10 microg x kg(-1) x h(-1) . Six pigs were given melagatran during the first 3 h of the 6-h endotoxaemic period . Nine controls were given the corresponding volume of saline instead of melagatran . Specimens from the liver and the left lung were taken for light microscopy post mortem . RESULTS: The endotoxin-induced increase in pulmonary capillary wedge pressure and drop in platelet count were significantly less pronounced in the melagatran-treated pigs . Deposits of pulmonary fibrin were significantly reduced in the melagatran group, without improving oxygenation . Light microscopy revealed no hepatic fibrin in the pigs treated with melagatran in contrast to the endotoxaemic controls . Hepatic neutrophil accumulation was reduced in the melagatran group as compared to controls . Hepatocellular degeneration and plasma levels of tumour necrosis factor alpha (TNF alpha) and bilirubin were of the same magnitude in both groups . CONCLUSION: Melagatran reduced pulmonary capillary wedge pressure, a retrograde reflection of the left ventricular end-diastolic filling pressure, and also pulmonary stasis in pigs subjected to endotoxaemic challenge . Pulmonary and hepatic fibrin depositions were reduced, but PaO2 levels or liver function markers were not affected by melagatran during the early phase of endotoxaemia . Obstruction of the intrahepatic bile ducts, by fibrin depositions, is not responsible for reduced excretion of conjugated bilirubin during endotoxaemia . The beneficial effects of melagatran during endotoxaemia were not due to any reduction of plasma TNF alpha.

Acta Anaesthesiol Scand, 2000 Jan, 44(1), 17 - 23
Lipid peroxidation induced by an early inflammatory response in endotoxaemia; Basu S et al.; BACKGROUND: Endotoxaemic challenge promptly causes lipid peroxidation . Porcine endotoxaemia can be used to replicate severe human septic shock . This model was used to evaluate non-enzymatic {8-Iso-prostaglandin F2alpha (8-Iso-PGF2alpha)} and enzymatic {15-keto-13,14-dihydro-prostaglandin F2alpha (15-K-DH-PGF2alpha)} lipid peroxidation, respectively, in relation to survival . The aim of this study was to correlate, if possible, pathophysiologic events during endotoxaemia to the levels of these arachidonic acid metabolites . METHODS: Nineteen pigs were anaesthetised, monitored (circulatory and respiratory variables in relation to lipid peroxidation) and given a continuous 6 h E . coli endotoxin (10 microg x kg(-1) x h(-1)) infusion . All animals were mechanically ventilated at constant tidal volumes and the inspired oxygen fraction was kept constant during the experimental period . RESULTS: This endotoxin infusion caused expressed derangements in all pigs and death in 9 of them . The levels of 8-Iso-PGF2alpha, indicating oxidative injury, were different in time course, magnitude and fashion between survivors and non-survivors . The levels of 15-K-DH-PGF2alpha, indicating inflammatory response, showed a similar pattern . At 1 h the CO2 partial pressure in arterial blood was significantly higher in non-surviving pigs and correlated (r: 0.7; P<0.05) to the levels of 15-K-DH-PGF2alpha . Prostaglandin F2alpha is mainly metabolised in the lung . The lung weights were significantly (P<0.05) higher in non-surviving than in surviving animals . Both free radical and cyclooxygenase catalysed oxidative modification occurs during endotoxaemia . CONCLUSION: Increased metabolism and inflammation, as evaluated by 15-K-DH-PGF2alpha, in the group of non-survivors may mediate the increase in arterial CO2 . Thus, increased lipid peroxidation seems to be associated with endotoxaemic organ dysfunction and increased mortality.

Arch Oral Biol, 2000 Jan, 45(1), 41 - 52
The molecular cloning, nucleotide sequence and expression of an antigenic determinant from Porphyromonas gingivalis; Rigg GP et al.; A genomic library generated in Escherichia coli was probed with a monoclonal antibody (mAb) LDS28, which reacts with a species-specific cell-surface antigen of Porphyromonas gingivalis . A clone designated pGPR2.1 was shown to express a 46-kDa protein reactive with mAb LDS28, which maps to a 1.7-kb HincII fragment . DNA sequence analysis revealed pGPR2.1 contains a 5653-bp insert with six open reading frames, one of which shows significant DNA homology with the rnhB gene of E . coli . Several subclones of pGPR2.1 were randomly generated in plasmid vector pTTQ18* using restriction enzyme Sau3a . Immunoblotting of subclones demonstrated that the LDS28-reactive antigen was coded for by an open reading frame predicted to specify a protein of 455 amino acids (50 kDa) . This open reading frame was designated pgaA (Porphyromonas gingivalis antigen) . The predicted amino acid sequence of PgaA contains a putative ABC signature for binding NTPs as well as a predicted transmembrane domain . Minicell labelling of pGPR2.1-encoded proteins and subclone derivatives revealed that pgaA directs expression of protein of multiple molecular weights (31-46 kDa) from its own promoter in E . coli, and that some of these forms may be caused by proteolysis of a 50-kDa precursor which itself shows a reduced apparent molecular weight (46 kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Toxicon, 2000 Jan, 38(1), 113 - 21
Induction of neutralizing antibodies against Tityus serrulatus toxins by immunization with a recombinant nontoxic protein; Guatimosim SC et al.; An immunogenic nontoxic protein (TsNTxP) was purified from the venom of the scorpion Tityus serrulatus (Ts) . This peptide is composed of 63 amino acid residues with a high degree of structural homology with the toxins isolated from Ts . The nucleotide sequence of the gene that encodes TsNTxP was obtained and also showed a high degree of similarity with genes encoding Tityus toxins {Guatimosim, S.C.F., Prado, V.F., Diniz, C.R., Chavez-Olortegui, C. . Kalapothakis, E., 1999 . Molecular cloning and genomic analysis of TsNTxP; an immunogenic protein from Tityus serrulatus scorpion venom . Toxicon 37, 507-517} . In the present study the TsNTxP gene was expressed in E . coli BL21DE3 cells as a fusion protein with maltose-binding protein . The recombinant protein (TsNTxPrec) was purified by affinity chromatography and used as an immunogen in rabbits . The antigenic specificity of anti-TsNTxPrec antibodies was compared by an enzyme-linked immunosorbent assay using TsNTxP, TstFG50 (the fraction of Ts venom that represents most of the toxicity of the crude venom) and the crude venom, to coat microtitration plates . Anti-TsNTxPrec antibodies had a comparable high cross-reactivity for all antigens tested . Concentrations of Ts venom equivalent to 20 LD50 were effectively neutralized by 1 ml of the anti-TsNTxPrec serum . This result provides basic data for the use of such recombinant scorpion protein as an immunogen in the development of antivenoms for clinical use.

Biochem Cell Biol, 1999, 77(6), 507 - 13
Identification of the Escherichia coli enzyme I binding site in histidine-containing protein, HPr, by the effects of mutagenesis; Brokx SJ et al.; The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR . Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al . 1999) . Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement . Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding . Except for mutations to His15 and Arg17, very little or no change in Vmax was found . Alanine replacements for Gln21, Thr52, and Leu55 have no effect . The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR . The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (>5-fold) indicating these residues have more significant roles in binding to enzyme I . Acidic replacement at Ser46 caused very large increases (>100-fold), while Gln51Glu gave a 3-fold increase in Km . While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.

Chin J Biotechnol, 1999, 15(1), 23 - 8
High level expression of the deleted and mutated urokinase-scFv fusion gene in Escherichia coli; Wang X et al.; The fusion gene of a specific anti-human fibrinogen D-dimer scFv and low molecular weight single chain urokinase (scu-PA-32K) was restricted, spliced, and digested with exonuclease Bal31 to obtain a series of deletion mutants . Study of their expressions in E.coli revealed that the key sequence which reduced its expression level resided in the fragment from 841 to 851 bp, in which a tandem of AGG codons (encoding arginine, rarely used in E.coli) existed . By means of PCR mediated site-directed mutagenesis, we altered these two AGG codons to CGT codons, which could be more efficiently translated in E.coli, and the expression level turned out to be about 30% of the total bacterial proteins while that of the natural gene was only 2%-3%.

Chin J Biotechnol, 1999, 15(1), 7 - 13
Cloning of Schistosoma japonicum Chinese strain 22.6kD membrane-associated protein (Sj-22.6) gene and its overproduction on Escherichia coli; Cai X et al.; A 567bp DNA fragment was amplified from Schistosoma japonicum adult worm mRNA by RT-PCR . Sequence analysis revealed that this fragment contained S . Japonicum Chinese strain membrane-associated protein (Sj-22.6) gene . Then this gene was cloned into the expression vector pGEX-4T, and subsequently expressed in Escherichia coli . The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography . Its molecular weight was about 48 kD . The yield of expression was around 40 mg/L E.coli culture . The immunological test suggested that the recombinant protein had good antigenity which could make a good basis for the research of its immunological function in Schistosomiasis.

Horm Metab Res, 1999 Dec, 31(12), 686 - 91
Detection of autoantibodies to the diabetes-associated antigen IA-2 by a sensitive enzyme-linked immunosorbent assay; Lobner K et al.; The tyrosine phosphatase like protein IA-2 is an important autoantigen in insulin-dependent diabetes mellitus (type 1 diabetes) . Autoantibodies to IA-2 (IA-2A) are present in the serum of patients with type 1 diabetes even before the onset of the disease . Previously, we reported on a radioimmune assay to detect IA-2A, using E . coli-derived 125I-labelled IA-2 as antigen . Although this assay could be shown to be equivalent to the common reference method for IA-2A detection (radioligand assays using in vitro synthesised 35S-methionine labelled antigen), the disadvantages of both assays with respect to synthesis and handling of the radioactive antigen limit their use in routine laboratories . In this study, we have evaluated a non-radioactive enzyme-linked immunosorbent assay (ELISA) for the simple detection of IA-2A . We report on an ELISA where the biotinylated intracytoplasmic part of IA-2 (IA-2ic) is captured on streptavidin-coated plates . The sensitivity of the ELISA was similar to the validated radioligand assay, as it detected 47 of 69 (69%) patients with type 1 diabetes as compared to 46 of 68 (67 %) with the reference method for IA-2A detection (radioligand assays using in vitro synthesised 35S-methionine labelled antigen) . Only 2 of 50 (4%) patients with autoimmune thyroid disease and 1 of 114 (1 %) healthy controls were detected in the ELISA, confirming specificity . There was a significant correlation between the ELISA and the radioligand assay (r = 0.64, p<0.001) . We conclude that this ELISA is suitable to detect IA-2A in the serum of patients with type 1 diabetes with a similar sensitivity and specificity to the radioligand assay . This ELISA will allow rapid and simple measurement of IA-2A where the radioligand assay is inconvenient or not available.

Nature, 2000 Jan 27, 403(6768), 444 - 7
Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio; Ryu WS et al.; Rotation of the bacterial flagellar motor is driven by an ensemble of torque-generating units containing the proteins MotA and MotB . Here, by inducing expression of MotA in motA- cells under conditions of low viscous load, we show that the limiting speed of the motor is independent of the number of units: at vanishing load, one unit turns the motor as rapidly as many . This result indicates that each unit may remain attached to the rotor for most of its mechanochemical cycle, that is, that it has a high duty ratio . Thus, torque generators behave more like kinesin, the protein that moves vesicles along microtubules, than myosin, the protein that powers muscle . However, their translation rates, stepping frequencies and power outputs are much higher, being greater than 30 microm s(-1), 12 kHz and 1.5 x 10(5) pN nm s(-1), respectively.

Nature, 2000 Jan 27, 403(6768), 439 - 44
Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein; GrandPre T et al.; Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS) . However, many classes of CNS axons can extend for long distances in peripheral nerve grafts . A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth . Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described . The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury . Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A . Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum . A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones . In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration . These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.

Cancer Res, 2000 Jan 15, 60(2), 266 - 8
Prostate mutations in rats induced by the suspected human carcinogen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine; Stuart GR et al.; Male lacl transgenic rats were fed a diet containing 200 ppm of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP), a heterocyclic amine present in cooked meats . PhIP was found to be a powerful prostate mutagen . After 61 days of treatment, the lacl mutant frequency was 71 x 10(-5), >20-fold higher than the spontaneous mutant frequency of 3.2 x 10(-5) . The predominant PhIP-induced mutations were G:C->T:A transversions and deletions of G:C bp . The results directly link PhIP-induced mutations with the earlier observation of PhIP-induced prostate cancer in rats and suggest that exposure to dietary PhIP could be a risk factor in the incidence of human prostate cancer.

Crit Care Med, 2000 Jan, 28(1), 202 - 8
Partial liquid ventilation with perflubron attenuates in vivo oxidative damage to proteins and lipids; Rotta AT et al.; OBJECTIVE: To determine the impact of partial liquid ventilation on the degree of pulmonary damage by reactive oxygen species in a model of acute lung injury caused by systemic endotoxemia . DESIGN: A prospective, controlled, in vivo, animal laboratory study . SETTING: Animal research facility of a health sciences university . SUBJECTS: Forty New Zealand White rabbits . INTERVENTIONS: Mature rabbits were anesthetized and instrumented with a tracheostomy and vascular catheters . Animals were assigned to receive either partial liquid ventilation (n = 16) with perflubron (18 mL/kg via endotracheal tube) or conventional mechanical ventilation (n = 16) . Both groups were ventilated using similar strategies, with an Fio2 of 1.0 and tidal volume as required to obtain a normal Paco2 . Animals were then given 0.9 mg/kg Escherichia coli endotoxin intravenously over 30 mins . Eight uninjured instrumented and mechanically ventilated animals served as controls . Partial liquid ventilation or conventional ventilation was continued for 4 hrs before the animals were killed . Lung homogenates were analyzed for malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE) concentrations using a colorimetric assay . To assess protein oxidative damage, carbonyl groups in protein side chains were derivatized with 2,4-dinitrophenylhydrazine followed by Western blotting with a dinitrophenylated-specific primary antibody . MEASUREMENTS AND MAIN RESULTS: MDA (713.42+/-662 vs . 1601.4+/-1156 nmol/g protein; p = .023) and MDA plus 4-HNE (1480.24+/-788 vs . 2675.2+/-1628 nmol/g protein; p = .038) concentrations were lower in animals treated with partial liquid ventilation compared with conventionally ventilated animals, respectively . Animals treated with partial liquid ventilation exhibited attenuation of dinitrophenylated-derivatized protein bands by Western blotting, indicating a reduction in protein oxidative damage . The presence of perfluorocarbon did not interfere with the MDA assay when assessed by independent analysis in vitro . Perflubron did not serve as a sink for peroxyl radicals produced in the aqueous phase during separate in vitro oxidation experiments . CONCLUSIONS: Partial liquid ventilation attenuates oxidative damage to lipids and proteins during experimental acute lung injury . This finding is not caused by binding of lipid peroxidation products to perflubron or by the peroxyl radical scavenging properties of perflubron.

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 92 - 4
Crystallization and preliminary X-ray analysis of the catalytic core of the alkylhydroperoxide reductase component AhpF from Escherichia coli; Bieger B et al.; Alkylhydroperoxide reductases (AhpR, E.C . 1.6.4.x) are essential for the oxygen tolerance of aerobic organisms, converting otherwise toxic hydroperoxides of lipids or nucleic acids to their corresponding alcohols . The AhpF component (521 amino-acid residues, 56.2 kDa) belongs to the family of pyridine nucleotide-disulfide oxidoreductases and channels electrons from NAD(P)H via a series of disulfides towards the AhpC component, which finally reduces the hydro-peroxide substrates . Crystals of the proteolytically truncated AhpF component (residues Asn208-Ala521) of the alkyl hydroperoxide reductase from Escherichia coli were grown under oxidizing conditions . The crystals belong to space group P3(2)21, with unit-cell parameters a = 60.4, c = 171.8 A . X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation . A molecular-replacement solution was found using the structure of thioredoxin reductase from Arabidopsis thaliana as a search model.

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 89 - 91
Crystallization and preliminary X-ray diffraction analysis of the NAD-dependent non--phosphorylating GAPDH of the hyperthermophilic archaeon Thermoproteus tenax; Brunner NA et al.; Recombinant non-phosphorylating NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic crenarchaeote Thermoproteus tenax has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion technique . Crystals of different habits were obtained from several precipitant solutions (salts and polyethylene glycols) . Preliminary X-ray analysis was performed with crystals grown in ammonium formate, which belonged to the primitive hexagonal space group P622, and had unit-cell parameters a = b = 184.8, c = 133.0 A, gamma = 120 degrees . Assuming a molecular weight of 55 kDa, a Matthews parameter of 3.3 A(3) Da(-1) is calculated assuming two molecules per asymmetric unit . The diffraction limit of these crystals is 2.5 A resolution.

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 86 - 8
Crystallization of the NADP-dependent beta-keto acyl-carrier protein reductase from Brassica napus; Fisher M et al.; The NADP-dependent beta-keto acyl-carrier protein reductase (BKR) from Brassica napus has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol of average molecular weight 1500 as the precipitant . The crystals belong to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 129 . 9, c = 93.1 A, alpha = beta = 90, gamma = 120 degrees . Calculated values for V(m), the use of rotation and translation functions and consideration of the packing suggest that the asymmetric unit contains a monomer . The crystals diffract to beyond 2.8 A resolution and are more amenable to X-ray diffraction analysis than those reported previously for the Escherichia coli enzyme . The structure determination of B . napus BKR will provide important insights into the catalytic mechanism of the enzyme and into the evolution of the fatty-acid elongation cycle by comparisons with the other oxidoreductase of the pathway, enoyl acyl-carrier protein reductase (ENR).

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 84 - 5
Crystallization and preliminary crystallographic studies of ribosome recycling factor from Escherichia coli; Yun J et al.; Ribosome recycling factor (RRF) catalyzes the disassembly of a termination complex during the final stage of protein synthesis . RRF from Escherichia coli has been crystallized with PEG 400 as precipitant at 287 K . The crystal belongs to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 48.08, c = 141.67 A . Native data were collected from a frozen crystal to a resolution of 3.0 A on a Cu Kalpha rotating-anode X-ray source.

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 81 - 3
Lactate dehydrogenase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray analysis; Lee BI et al.; L(+)-Lactate dehydrogenase (LDH) is a key enzyme in anaerobic metabolism which converts pyruvate to lactate . LDH from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized in two crystal forms at 297 K using 2-methyl-2,4-pentanediol as precipitant . Type I crystals grew rapidly and diffracted to at least 2.8 A Bragg spacing upon exposure to Cu Kalpha X-rays . X-ray diffraction data to 2.9 A have been collected from a native crystal . The type I crystal is tetragonal, belonging to the space group P4(2)2(1)2, with unit-cell parameters a = b = 99.74, c = 170.00 A . The asymmetric unit contains two LDH subunits, with a corresponding crystal volume per protein mass (V(m)) of 3.05 A(3) Da(-1) and a solvent content of 59.7% . Type II crystals, which grew more slowly, diffracted to at least 1.8 A Bragg spacing upon exposure to Cu Kalpha X-rays . X-ray diffraction data to 1.9 A have been collected from a native crystal . The type II crystal is orthorhombic, belonging to the space group P2(1)2(1)2, with unit-cell parameters a = 47.65, b = 125.10, c = 58.08 A . The asymmetric unit contains a single LDH subunit, with a corresponding crystal volume per protein mass (V(m)) of 2.50 A(3) Da(-1) and a solvent content of 50.8% . Therefore, the type II crystal is more suitable for high-resolution structure determination than the type I crystal.

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 76 - 8
Toxoplasma gondii adenosine kinase: expression, purification, characterization, crystallization and preliminary crystallographic analysis; Recacha R et al.; The obligate intracellular protozoan parasite Toxoplasma gondii depends on the purine-salvage pathway for its purine supply . Unlike its mammalian hosts, T . gondii salvages purine precursors predominantly via adenosine kinase, the enzyme that phosphorylates adenosine to adenosine monophosphate (AMP) . The cDNA encoding T . gondii adenosine kinase was subcloned and expressed in Escherichia coli . The recombinant protein was active in an in vitro enzyme assay over a broad pH range . It required a divalent cation for activity . The enzyme was inactivated by the addition of 1 microM mercuric chloride . The inactivation could be reversed by a reducing agent . The active recombinant protein was crystallized using sodium sulfate as precipitant at pH 8.0 . The crystals diffract to 1.8 A and belong to the monoclinic space group P2(1), with unit-cell parameters a = 47.5, b = 68.9, c = 57.0 A, beta = 100.3 degrees . The calculated V(m) based on one molecule per asymmetric unit is 2.38 A(3) Da(-1).

Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 64 - 6
Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance-B (NusB) from Mycobacterium tuberculosis; Gopal B et al.; N-utilizing substance B (NusB) is a protein which forms part of a complex assembly in transcriptional antitermination in Mycobacterium tuberculosis . It forms a heterodimer with the product of the NusE gene (identical to the ribosomal protein S10) and mediates the process of transcriptional antitermination by forming the core complex with the nut site of the ribosomal RNA along with other protein factors . NusB has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method . The space group is P2(1)2(1)2(1), with unit-cell parameters a = 46.6, b = 64.2, c = 90.1 A . A native data set complete to 1.6 A resolution has been collected from a single crystal.

Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 212 - 4
Expression, crystallization and preliminary X-ray studies of the PDZ domain of Dishevelled protein; Khlebtsova N et al.; Dishevelled (Dsh) protein is an important component of the Wnt signal-transduction pathway . It has three relatively conserved domains: DIX, PDZ and DEP . The PDZ domain of the Xenopus laevis homolog of Dsh, which consists of residues 254-348, was overexpressed as a soluble protein in Escherichia coli, purified and crystallized . The crystals were obtained by the vapor-diffusion method, using 1.4 M sodium formate as a precipitant . The crystals diffracted to 2.3 A resolution . The space group was determined to be P6(1)22 or P6(5)22, with unit-cell dimensions a = b = 95.9, c = 93.9 A.

Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 210 - 1
Crystallization and preliminary X-ray diffraction studies of a beta-carbonic anhydrase from the red alga Porphyridium purpureum; Mitsuhashi S et al.; The beta-carbonic anhydrase from the red alga Porphyridium purpureum was heterologously expressed, purified and crystallized . The crystals belong to space group P2(1) (unit-cell parameters a = 63.8, b = 113.9, c = 73.8 A, beta = 104.1 degrees) with two subunits per asymmetric unit and diffract to 2.5 A resolution.

Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 206 - 9
Crystallization and preliminary crystallographic analysis of phosphonoacetaldehyde hydrolase; Morais MC et al.; Phosphonoacetaldehyde hydrolase, a C-P bond-cleaving enzyme which utilizes an unusual bicovalent catalytic strategy, has been crystallized by the hanging-drop vapor-diffusion method using PEG 4000 as the precipitant . The crystals belong to the monoclinic system and belong to space group C2, with unit-cell parameters a = 210.5, b = 45.5, c = 64.7 A, beta = 105.0 degrees . The asymmetric unit contains a dimer related by a non-crystallographic dyad . In addition to a 2.7 A native data set, the following data sets have been collected: a 2.4 A data set from crystals complexed with the intermediate analog vinyl sulfonate, a 3.0 A three-wavelength MAD data set from crystals complexed with the product analog WO(4)(2-), as well as several heavy-atom data sets to 3.0 A or better, of which only three have proven useful for MIR calculations . Examination of the native Patterson map revealed NCS that made previously uninterpretable derivative data useful . Independent phase sets were first calculated and refined for the MAD and MIR experiments separately and were then combined . The combined phase set was further improved by solvent flattening, histogram matching and NCS averaging . Interpretation of the resulting electron-density map is currently under way.

Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 187 - 8
Crystallization and preliminary X-ray crystallographic analysis of the 30 kDa membrane-binding domain of protein 4.1 from human erythrocytes; Han BG et al.; The 30 kDa membrane-binding domain of protein 4.1 from human erythrocytes has been expressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study . Crystals were grown using a salting-in technique . Crystals have a tetragonal plate shape and belong to the C2 space group, with unit-cell parameters a = 163.9, b = 106.5, c = 93.5 A, beta = 95.5 degrees . The crystals diffract to 2.8 A resolution.

Acta Crystallogr D Biol Crystallogr, 1999 Dec, 55 ( Pt 12), 2053 - 5
Crystallization of the N-terminal domain of human sex hormone-binding globulin, the major sex steroid carrier in blood; Grishkovskaya I et al.; The amino-teminal laminin G-like domain of human sex hormone-binding globulin (SHBG), which contains the steroid-binding site and the dimerization domain, has been produced in Escherichia coli, purified to homogeneity and crystallized in complex with 5alpha--dihydrotestosterone (DHT) in two different crystal forms . Native data sets have been collected for tetragonal crystals (space group P4(1)22 or P4(3)22; unit-cell parameters a = 52.2, c = 148.4 A) diffracting to 3.3 A and trigonal crystals (R32; a = 104.0, c = 84.4 A) diffracting to better than 1.6 A . Since both crystal forms can only accommodate a single monomer in the asymmetric unit and share twofold rotational symmetry, it is proposed that the homodimer of this truncated form of SHBG, as observed in ultracentrifugation experiments, displays C(2) point-group symmetry.

Acta Crystallogr D Biol Crystallogr, 1999 Dec, 55 ( Pt 12), 2033 - 4
Crystallization and preliminary X-ray analysis of the Escherichia coli UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (MurF); Yan Y et al.; Crystals of the Escherichia coli UDP-MurNAc-tripeptide D-Ala-D-Ala-adding protein (MurF), which catalyzes the formation of the last metabolite of the bacterial cell-wall building block, have been grown in hanging-drop vapor-diffusion trials using PEG 8K as a precipitating agent . The crystals belong to hexagonal space group P6(1) or P6(5), with unit-cell dimensions a = b = 74, c = 425 A . The asymmetric unit contains two molecules, with a crystal volume per protein mass (V(m)) of 3.4 A(3) Da(-1) and a solvent content of about 64% by volume . A native data set to 2.8 A resolution has been obtained from a frozen crystal using a synchrotron X-ray source.

Acta Crystallogr D Biol Crystallogr, 1999 Dec, 55 ( Pt 12), 2028 - 30
Expression, purification, crystallization and preliminary X-ray analysis of Escherichia coli argininosuccinate synthetase; Lemke C et al.; A recombinant form of Escherichia coli argininosuccinate synthetase with a C-terminal polyhistidine affinity tag has been expressed, purified and subsequently crystallized using the hanging-drop vapour-diffusion technique . The crystals grow as large rectangular chunks with unit-cell dimensions a = 79.70, b = 105.84, c = 127.33 A, alpha = beta = gamma = 90 degrees . The crystals exhibit the symmetry of space group I222 and diffract to a minimum d-spacing of 1.6 A at station X8C of the National Synchrotron Light Source, Brookhaven National Laboratory . On the basis of density calculations, one monomer of this homotetrameric protein is predicted per asymmetric unit (Matthews coefficient V(m) = 2.69 A(3) Da(-1)).

Arch Biochem Biophys, 2000 Feb 15, 374(2), 381 - 8
Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase; Laden BP et al.; The cDNA for human squalene monooxygenase, a key enzyme in the committed pathway for cholesterol biosynthesis, was amplified from a human liver cDNA library and cloned, and the protein was expressed in Escherichia coli and purified . Kinetic analysis of the purified enzyme revealed an apparent K(m) for squalene of 7.7 microM and an apparent k(cat) of 1.1 min(-1) . For FAD the apparent K(m) is 0.3 microM, consistent with a loosely bound flavin . The apparent K(m) for NADPH-cytochrome P450 reductase, the requisite electron transfer partner, is 14 nM . The amount of reductase needed for maximal activity is about threefold less than the amount of squalene monooxygenase present in the assay; thus, electron transfer to the monooxygenase is not likely to be rate limiting . Previous reports have implicated inhibition of this enzyme as the cause of a peripheral demyelination seen in weanling rats fed a diet containing tellurium . As no data were available for humans, the ability of a number of tellurium and related elemental compounds to inhibit the recombinant human enzyme was examined . Tellurite, tellurium dioxide, selenite, and selenium dioxide were inhibitory; the tellurium compounds were more potent than the selenium compounds, as indicated by their IC(50) values (17 and 37 microM, respectively) . Kinetic analysis of the inhibition by tellurite suggests multiple sites of interaction with the enzyme in a noncompetitive manner with respect to squalene .

Arch Biochem Biophys, 2000 Feb 15, 374(2), 371 - 80
Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli; Walker K et al.; The taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonate-induced Taxus cells, and partially purified and characterized (K . Walker, R . E . B . Ketchum, M . Hezari, D . Gatfield, M . Golenowski, A . Barthol, and R . Croteau, Arch . Biochem . Biophys . 364, 273-279 1999) . A revised purification method allowed internal amino acid microsequencing of the enzyme, from which primers were designed and employed to amplify a transacetylase gene-specific fragment . This radiolabeled, 900-bp amplicon was used as a hybridization probe to screen a cDNA library constructed from poly(A)(+) RNA isolated from induced Taxus cells, from which a full-length transacetylase sequence was obtained . Expression of this clone from pCWori(+) in Escherichia coli JM109 cells yielded the functional enzyme, as determined by radiochemical assay and combined capillary gas chromatographic-mass spectrometric verification of the acetylated product . The full-length DNA has an open-reading frame of 1317 nucleotides corresponding to a deduced amino acid sequence of 439 residues that exhibits high sequence identity to the proteolytic fragments of the native enzyme, which the recombinant transacetylase resembles in properties . Consistent with the size of the operationally soluble native enzyme, the DNA appears to encode a monomeric protein of molecular weight 49,079 that bears no N-terminal organellar targeting information . Sequence comparison of the taxadien-5alpha-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (64-67%) . The efficient conversion of taxadien-5alpha-yl acetate to further hydroxylated intermediates of the Taxol pathway confirms the significance of this acylation step and suggests this taxadienol transacetylase to be an important target for genetic manipulation to improve Taxol production .

Arch Biochem Biophys, 2000 Feb 15, 374(2), 261 - 8
Cloning and characterization of glyoxalase I from soybean; Skipsey M et al.; Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments . In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No . X68819) resembling a GST being described as a glyoxalase I . To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures . When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions . GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or gamma-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean . When the clone X68819 was expressed in E . coli, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3 . GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene . Both enzymes are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%) .

Nucleic Acids Res . 2000 Mar 1;28(5):E14.
Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli; Yue BG et al.; Mammalian proteins expressed in Escherichia coli are used in a variety of applications . A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s) . Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate . We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei . The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.

Nucleic Acids Res, 2000 Mar 1, 28(5), 1245 - 51
Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli; Monteilhet C et al.; The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs . Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain . It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing . Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli . We have also determined the cleavage pattern as well as the recognition site of p28bi2 . It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs . Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.

Nucleic Acids Res, 2000 Mar 1, 28(5), 1176 - 82
Specific bonding of puromycin to full-length protein at the C-terminus; Miyamoto-Sato E et al.; Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains . Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g . 0.04 microM) can bond only to full-length protein at the C-terminus . This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives . The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon . The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R . Puromycin and its derivatives at 0 . 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates . Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors . These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon . This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

Nucleic Acids Res, 2000 Mar 1, 28(5), 1170 - 5
Stereochemical control of DNA biosynthesis; Sosunov VV et al.; Stereochemical control of DNA biosynthesis was studied using several DNA-synthesizing complexes containing, in each case, a single substitution of a 2'-deoxy-D-nucleotide residue by an enantiomeric L-nucleotide residue in a DNA chain (either in the primer or in the template) as well as 2'-deoxy-L-ribonucleoside 5'-triphosphates (L-dNTPs) as substrates . Three template-dependent DNA polymerases were tested, Escherichia coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase and avian myeloblastosis virus reverse transcriptase, as well as template-independent calf-thymus terminal deoxynucleotidyl transferase . Very stringent control of stereoselectivity was demonstrated for template-dependent DNA polymerases, whereas terminal deoxynucleotidyl transferase was less selective . DNA polymerase I and reverse transcriptase catalyzed formation of dinucleoside 5',5'-tetraphosphates when L-dTTP was used as substrate . Comparison between models of template-primer complexes, modified or not by a single L-nucleotide residue, revealed striking differences in their geometry.

Nucleic Acids Res, 2000 Mar 1, 28(5), 1085 - 91
Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking; Kubareva EA et al.; A target sequence-specific DNA binding region of the restriction endonuclease Sso II was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-IdU) at the central position of the Sso II recognition site (CCNGG) . For this purpose the Sso II-DNA complex was irradiated with a helium/cadmium laser (325 nm) . The cross-linking yield obtained was approximately 50% . In the presence of excess unmodified oligodeoxynucleotide or with oligode-oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction . The cross-linked Sso II-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxy-nucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61 . In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage . It is concluded that the region around Trp61 is involved in specific binding of Sso II to its DNA substrate.

Nucleic Acids Res, 2000 Mar 1, 28(5), 1045 - 52
A novel RNA-binding protein from Triturus carnifex identified by RNA-ligand screening with the newt hammerhead ribozyme; Denti MA et al.; The newt hammerhead ribozyme is transcribed from Satellite 2 DNA, which consists of tandemly repeated units of 330 bp . However, different transcripts are synthesized in different tissues . In all somatic tissues and in testes, dimeric and multimeric RNA transcripts are generated which, to some extent, self-cleave into monomers at the hammerhead domain . In ovaries, primarily a distinct monomeric unit is formed by transcription, which retains an intact hammerhead self-cleavage site . The ovarian monomeric RNA associates to form a 12S complex with proteins that are poorly characterised so far . In this work we identified NORA, a protein that binds the ovarian form of the newt ribozyme . We show that the newt ribozyme binds to the Escherichia coli -expressed protein, as well as to a protein of identical size that is found exclusively in newt ovaries . Also NORA mRNA was detectable only in ovary, but in neither somatic tissues nor testes . The tissue-specific expression of NORA is analogous to the ovary-specific transcription of the newt ribozyme . Although NORA was identified by its ability to bind to the newt ribozyme in the presence of a vast excess of carrier RNA, it was able to interact with certain other RNA probes . This novel RNA-binding protein does not contain any motif characteristic for RNA-binding proteins or any other known protein domain, but it shares a striking similarity with a rat resiniferatoxin-binding protein.

J Virol, 2000 Mar, 74(5), 2186 - 92
Characterization of human CD4(+) T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein; ter Meulen J et al.; T cells must play the major role in controlling acute human Lassa virus infection, because patients recover from acute Lassa fever in the absence of a measurable neutralizing antibody response . T cells alone seem to protect animals from a lethal Lassa virus challenge, because after experimental vaccination no neutralizing antibodies are detectable . In order to study human T-cell reactivity to single Lassa virus proteins, the nucleoprotein (NP) of Lassa virus, strain Josiah, was cloned, expressed in Escherichia coli, and affinity purified . Peripheral blood mononuclear cells (PBMC) obtained from 8 of 13 healthy, Lassa virus antibody-positive individuals living in the Republic of Guinea, western Africa, were found to proliferate in response to the recombinant protein (proliferation index >/=10) . PBMC obtained from one individual with a particularly high proliferative response were used to generate 50 NP-specific T-cell clones (TCC) . For six of these the epitopes were mapped with overlapping synthetic peptides derived from the sequence of the NP . These CD4(+) TCC displayed high specific proliferation and produced mainly gamma interferon upon stimulation with NP . Because variation of up to 15% in the amino acid sequences of the structural proteins of naturally occurring Lassa virus variants has been observed, the reactivity of the TCC with peptides derived from the homologous epitopes of the Nigeria strain of Lassa virus and of the eastern Africa arenavirus Mopeia was tested . With the Nigeria strain of Lassa virus the levels of homology were 100% for two of these epitopes and 85% for three of them, whereas homology with the respective Mopeia epitopes ranged from 92 to 69% . Reactivity of the TCC with peptides derived from the variable epitopes of the Nigeria strain and of Mopeia was reduced or completely abolished . This report shows for the first time that seropositive individuals from areas of endemicity have very strong memory CD4(+) T-cell responses against the NP of Lassa virus, which are partly strain specific and partly cross-reactive with other Lassa virus strains . Our findings may have important implications for the strategy of designing recombinant vaccines against this mainly T-cell-controlled human arenavirus infection.

J Virol, 2000 Mar, 74(5), 2057 - 66
Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus; Petit S et al.; The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3 . Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity . Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions . Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679 . A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine . Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679 . The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases . Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis . Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis . The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

Blood, 2000 Feb 15, 95(4), 1342 - 9
Stimulation of cytotoxic T cells against idiotype immunoglobulin of malignant lymphoma with protein-pulsed or idiotype-transduced dendritic cells; Osterroth F et al.; Because of their hypervariable regions and somatic mutations, the antigen receptor molecules of lymphomas (idiotypes) are tumor-specific antigens and attractive targets for antilymphoma immunotherapy . For the optimal induction of human idiotype-specific cytotoxic T cells (CTL), idiotype was presented to CD8(+) peripheral blood mononuclear cells by monocyte-derived autologous dendritic cells (DC) after the endocytosis of idiotype protein or by idiotype-expressing DC . Recombinant idiotype was obtained as a functionally folded Fab fragment by periplasmic expression in Escherichia coli . Idiotype-expressing DC were generated by transduction with recombinant Semliki forest virus vectors encompassing heavy- or light-chain idiotype genes . Autologous lymphoblastoid cell lines stably transfected with Epstein-Barr virus-based idiotype expression vectors were used as target cells to detect idiotype-specific lysis . CTL stimulated with idiotype-loaded DC showed strong specific, CD8-mediated, and major histocompatibility complex (MHC) class I-restricted cytotoxicity against autologous heavy- and light-chain idiotype . In contrast, stimulation with idiotype-transduced DC resulted in only moderate natural killer cell activity . These data confirm the existence of idiotype-specific CTL in patients with lymphoma, define a "good manufacturing practice"-compatible protocol for the generation of these cells without the requirement of viable lymphoma cells, and favor the processing of exogenous antigen over DC transduction for the induction of MHC I-restricted CTL against idiotypes with unknown antigenicity . (Blood . 2000;95:1342-1349)

Blood, 2000 Feb 15, 95(4), 1117 - 23
Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli; Minnema MC et al.; Plasma-derived antithrombin III (ATIII) prevents the lethal effects of Escherichia coli infusion in baboons, but the mechanisms behind this effect are not clear . In the present study, we evaluated the effects of recombinant human ATIII (rhATIII) on the clinical course and the inflammatory cytokine and coagulation responses in baboons challenged with lethal dose of E coli . Animals in the treatment group (n = 5) received high doses of rhATIII starting 1 hour before an E coli challenge . Those in the control group were administered saline . Survival was significantly improved in the treatment group (P =.002) . Both groups had similar hemodynamic responses to E coli challenge but different coagulation and inflammatory responses . The rhATIII group had an accelerated increase of thrombin-ATIII complexes and significantly less fibrinogen consumption compared to controls . In addition, the rhATIII group had much less severe thrombotic pathology on autopsy and virtually no fibrinolytic response to E coli challenge . Furthermore, the rhATIII group had a significantly attenuated inflammatory response as evidenced by marked reduction of the release of various cytokines . We conclude that the early administration of high doses of rhATIII improves the outcome in baboons lethally challenged with E coli, probably due to the combined anticoagulation and anti-inflammatory effects of this therapy . (Blood . 2000;95:1117-1123)

Am J Physiol Cell Physiol, 2000 Feb, 278(2), C336 - 43
Cyclic nucleotide-gated cation channels mediate sodium and calcium influx in rat colon; Qiu W et al.; We found mRNA for the three isoforms of the cyclic nucleotide-gated nonselective cation channel expressed in the mucosal layer of the rat intestine from the duodenum to the colon and in intestinal epithelial cell lines in culture . Because these channels are permeable to sodium and calcium and are stimulated by cGMP or cAMP, we measured 8-bromo-cGMP-stimulated sodium-mediated short-circuit current (I(sc)) in proximal and distal colon and unidirectional (45)Ca(2+) fluxes in proximal colon to determine whether these channels could mediate transepithelial sodium and calcium absorption across the colon . Sodium-mediated I(sc), stimulated by 8-bromo-cGMP, were inhibited by dichlorobenzamil and l-cis-diltiazem, blockers of cyclic nucleotide-gated cation channels, suggesting that these ion channels can mediate transepithelial sodium absorption . Sodium-mediated I(sc) and net transepithelial (45)Ca(2+) absorption were stimulated by heat-stable toxin from Escherichia coli that increases cGMP . Addition of l-cis-diltiazem inhibited the enhanced transepithelial absorption of both ions . These results suggest that cyclic nucleotide-gated cation channels simultaneously increase net sodium and calcium absorption in the colon of the rat.

Int J Radiat Biol, 2000 Jan, 76(1), 43 - 9
Histone-like protein HU is required for recA gene-dependent DNA repair and SOS induction pathways in UV-irradiated Escherichia coli; Miyabe I et al.; PURPOSE: Escherichia coli HU protein exists as a heterodimer composed of two highly homologous subunits, HU-1 and HU-2, encoded by the hupB and hupA genes, respectively . It introduces negative supercoils into a relaxed circular DNA . Various roles of HU have been suggested in cellular processes such as DNA replication and transcription . The present experiments were designed to understand the role of HU in DNA repair processes in E . coli . MATERIALS AND METHODS: The sensitivity of hupA/hupB mutants of E . coli to the lethal and mutagenic effects of UV was compared with that of a wild-type strain . The effect of the hupAhupB mutations in SOS induction was also examined . RESULTS: The hupAhupB mutations increased the UV sensitivity of E . coli . Nucleotide excision repair was unaffected by the deficiency of HU . On the other hand, E . coli hupAhupB mutants were sensitive to UV in the recA+recB+recF background but not in the recArecB+recF+ or recA+recBrecF+ background . The frequency of UV-induced mutation to rifampicin resistance was significantly reduced in the hupAhupB mutants, and the induction of the recA::lacZ and umuC::lacZ fusion genes was also suppressed in the mutants . CONCLUSIONS: HU protein plays a critical role in the recA, recB-dependent recombinational DNA repair and SOS induction pathways in UV-irradiated E . coli.

Int J Radiat Biol, 2000 Jan, 76(1), 1 - 9
Effect of ethidium bromide intercalation on DNA radiosensitivity; Begusova M et al.; PURPOSE: To assess the influence of the intercalating drug ethidium bromide (EtBr) on the yields of single strand breaks (ssb) induced by fast neutrons in supercoiled pBR322 plasmid and in a linear DNA restriction fragment . MATERIALS AND METHODS: The yield of ssb in the plasmid was measured by agarose gel electrophoresis . The proportion of fragments bearing one ssb and the probability of breakage at each nucleotide site was determined using sequencing gel electrophoresis . The volume variations due to the intercalation of EtBr were calculated . The expected radio-modifying effect at each nucleotide site of the linear fragment was evaluated using a reported simulation procedure . RESULTS: The ssb yield in the plasmid increased for concentrations up to 0.04 drug/bp and fell back in the range 0.04-0.1 drug/bp . For the linear DNA, only a slight protective effect was observed over the whole concentration range . The effect was almost the same at all nucleotide sites . CONCLUSION: For the linear DNA fragment, radioprotection was mainly due to scavenging of OH* radicals by the intercalated drug . For the plasmid, the radio-modifying effect results mainly from the variation of its effective volume, due to the modification of superhelicity.

Toxicon, 2000 Feb, 38(2), 153 - 62
Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider (Selenocosmia huwena); Li M et al.; Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider Selenocosmia huwena . A gene encoding huwentoxin-I, a peptide neurotoxin consisted of 33 amino acid residues from the venom of the Chinese bird spider Selenocosmia huwena, was designed, synthesized and expressed in Escherichia coli as a hybrid protein fused with glutathione S-transferase at the N-terminal . The fusion protein was purified by GSH-Sepharose 4B affinity column chromatography and cleaved by thrombin to release the toxin peptide . The amino acid sequence of the recombinant toxin was consistent with the designed one by sequence determination and MALDI-TOF mass analysis, suggesting that the recombinant huwentoxin-I produced the same expression product as the native one . After reduction and renaturation, the biological activity of the recombinant toxin was identical with that of the native huwentoxin-I by electrophysiological method.

Vet Microbiol, 2000 Jan, 71(1-2), 69 - 80
The association of two recombinant proteinases of a feline strain of Porphyromonas gingivalis with periodontal disease in cats; Norris JM et al.; Serum from 40 domestic cats with various grades of periodontal disease was used to probe two recombinant functional proteinases from feline strain VPB 3457 of Porphyromonas gingivalis expressed in E . coli . One recombinant proteinase (VPB 2856) was constructed using polymerase chain reaction and had 91% DNA identity with the prtC collagenase gene of the human type strain of P . gingivalis, while the other proteinase (VPB 2814) was isolated from a size selected genomic library and had an amino-terminal sequence with no significant identity with deposited sequences . Thirteen of 40 cats showed a serum antibody response to VPB 2856 using Western immunoblot detection . All the 13 cats had an overall periodontal grade of 3 or greater and greater than 1.68x10(5) cfu P . gingivalis at the canine and premolar periodontium sample sites . Fourteen of 40 cats showed a serum antibody response to VPB 2814 . Thirteen of these 14 cats had an overall periodontal grade of 3 or greater . Regression analysis of overall periodontal grade against the serum antibody response showed significant positive relationships for both VPB 2856 (r2 = 0.351; p<0.001) and VPB 2814 (r2 = 0.247; p<0.001) . Regression analysis of the total colony forming units of feline strain P . gingivalis against the grade of serum antibody response showed a positive relationship for both VPB 2856 (r2 = 0.662; p<0.001) and VPB 2814 (r2 = 0.531; p<0.001) . These data provide strong evidence that the recombinant proteinases of feline P . gingivalis expressed in E . coli clones VPB 2856 and VPB 2814 are associated with periodontal disease in cats.

Chemosphere, 2000 Feb, 40(4), 369 - 73
Degradation and toxicity reduction of textile effluent by combined photocatalytic and ozonation processes; de Moraes SG et al.; To minimize the environmental impact of textile effluents, mainly related to their high coloration and the presence of toxic or carcinogenic reactive dyes, the efficiency of photochemical and ozonation processes, applied in the form of isolated and combined procedures, were evaluated . The investigation was focused on the reduction of total organic carbon content (TOC), color and acute toxicity (monitoring by inhibition of Escherichia coli respiration) . For a reaction time of 60 min, the anatase TiO2-assisted photocatalytic process produces color and TOC reduction of about 90% and 50%, respectively . Meanwhile, the ozonation process gives a decolorization of about 60% but negligible TOC reduction . When the processes were applied in a simultaneous form, the decolorization was almost complete and the TOC reduction was higher than 60% . The three treatments studied yield an acute toxicity reduction of around 50%.

Yi Chuan Xue Bao, 1999, 26(5), 512 - 7
{Western blotting of RStV gene products in rice and insects}; Qu ZC et al.; The NS3 and NC protein genes encoded by RNA3 of RStV, the NCP and NSvc4 protein genes encoded by RNA4 were subcloned into the E . coli expression vector pGEX3X to express four groups of fusion protein under IPTG induction . These fusion proteins were used to immunize rabbits to raise antisera . The antisera against the E . coli-expressed proteins were available for probing the presence of the viral gene products in both rice plant and insect hosts . The expected gene products can be probed only in diseased rice plant with NCP antiserum and the corresponding products detected in both plant and RStV particle preparation with NC antiserum . The viral gene products probed by NS3 and NSvc4 antisera were different from the expected ones in size.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2248 - 51
Synthesis and characterization of intermediate and transition-state analogue inhibitors of gamma-glutamyl peptide ligases; Inoue M et al.; The phosphonodifluoromethyl ketone and phosphonofluoridate derivatives of L-glutamic acid were synthesized and characterized as analogues of the gamma-glutamyl phosphate intermediate and the tetrahedral transition state, respectively, for the inhibition of gamma-glutamylcysteine synthetase and glutamine synthetase . The former served as a poor inhibitor of both enzymes, but the latter inhibited glutamine synthetase with a Ki of 59 microM and partially inactivated the enzyme in an NH3- and ATP-dependent manner.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2157 - 62
Analysis of a catalytic acidic pair in the active center of cellulase from Aspergillus aculeatus; Ohnishi A et al.; Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the X-ray crystallographic analysis of FI-CMCase, endo-1,4-beta-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No . F-50 . To identify the catalytic residues of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes expressed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S . Mutant enzymes E118S and E202S were not shown to have any detectable activity . Kinetic parameters of other mutant enzymes were measured after purification . The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively . From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2123 - 9
Local antibody response in Peyer's patches to the orally administered dietary protein antigen; Shimoda M et al.; To understand local antibody production to dietary protein antigens in the gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer's patches of BALB/c mice raised against orally administered hen egg lysozyme (HEL) was studied . These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13 clones) isotypes . Some of the HEL-binding mAbs preferentially reacted with reduced, carboxy-methylated HEL, rather than with native HEL . MAbs of the IgA and IgM isotypes had cross-reactivity with other unrelated environmental antigens such as E . coli, single-strand DNA, and soluble components of mouse food . In contrast, the IgG1 mAbs did not cross-react with these antigens . The average of the Kd values for HEL of these mAbs was in the order of 10(-6) M, which is moderately higher than those of mAbs from the preimmune repertoire . These results suggest that, under normal physiological conditions, orally administered dietary proteins predominantly induce the local production of polyreactive IgA/IgM antibodies cross-reacting with environmental luminal antigens.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2069 - 74
Amino acid sequence analysis of bitter peptides from a soybean proglycinin subunit synthesized in Escherichia coli; Kim MR et al.; The cDNA encoding A1aB1b proglycinin was expressed in E . coli, for the efficient isolation of a single peptide responsible for the bitterness . The 55-kD proglycinin was highly purified, hydrolyzed, and further purified through a series of chromatographic steps to yield fractions with the major bitter peptides . The most bitter-tasting fractions contained peptides with average molecular weights lower than 1,700 Da . An analysis of the amino acid sequences indicated that many small bitter peptides (< 1,000 Da) are composed of uncharged polar amino acids as well as hydrophobic amino acids, with a charged residue often being present at either end . This suggests the involvement of a certain structural requirement in taste perception.

Trends Biochem Sci, 2000 Feb, 25(2), 39 - 43
A structure-based mechanism for drug binding by multidrug transporters; Zheleznova EE et al.; Multidrug transporters bind chemically dissimilar, potentially cytotoxic compounds and remove them from the cell . How these transporters carry out either of these functions is unknown . On the basis of crystal structures of the multidrug-binding domain of the transcription activator BmrR and mutagenesis studies on the bacterial multidrug transporter MdfA, we propose a possible mechanism for the binding of cationic lipophilic drugs by multidrug transporters . The key element of this mechanism includes a conformational change in the transporter that exposes a buried charged residue in the substrate-binding pocket and allows access to this site by only those drugs that are its steric and electrostatic complements.

FEBS Lett, 2000 Feb 4, 467(1), 105 - 10
Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH-2) and analysis of its interaction with ubiquinone analogues; Bjorklof K et al.; The NADH:ubiquinone reductase (NDH-2) of Escherichia coli was expressed as a His-tagged protein, extracted from the membrane fraction using detergent and purified by chromatography . The His-tagged NDH-2 was highly active and catalyzed NADH oxidation by ubiquinone-1 at rates over two orders of magnitude higher than previously reported . The purified, His-tagged NDH-2, like native NDH-2, did not oxidize deamino-NADH . Steady-state kinetics were used to analyze the enzyme's activity in the presence of different electron acceptors . High V(max) and low K(m) values were only found for hydrophobic ubiquinone analogues, particularly ubiquinone-2 . These findings strongly support the notion that NDH-2 is a membrane bound enzyme, despite the absence of predicted transmembrane segments in its primary structure . The latter observation is in agreement with possible evolutionary relation between NDH-2 and water-soluble enzymes such as dihydrolipoamide dehydrogenase . There is currently no clear indication of how NDH-2 binds to biological membranes.

FEBS Lett, 2000 Feb 4, 467(1), 97 - 100
Different lumen-targeting pathways for nuclear-encoded versus cyanobacterial/plastid-encoded Hcf136 proteins; Hynds PJ et al.; Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state . We present the first evidence that a given protein can be targeted by different pathways in different organisms . Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine . However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec) . We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway.

FEBS Lett, 2000 Feb 4, 467(1), 91 - 6
Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family; Vayssiere B et al.; Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure . Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP . We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7 . This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors . The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.

FEBS Lett, 2000 Feb 4, 467(1), 37 - 40
Efficient introduction of aryl bromide functionality into proteins in vivo; Sharma N et al.; Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials . The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means . In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity . Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog . Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe . The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions . In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.

FEBS Lett, 2000 Feb 4, 467(1), 17 - 21
Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim-1 kinase and the effect of phosphorylation on the transcriptional repression function of HP1(1); Koike N et al.; Pim-1, a protooncogene product, is a serine/threonine kinase and is thought to play a role in signal transduction in blood cells . Few phosphorylated target proteins for Pim-1, however, have been identified . In the present study, two-hybrid screening to clone cDNAs encoding proteins binding to Pim-1 was carried out, and a cDNA for heterochromatin protein 1gamma (HP1gamma) was obtained . Binding assays both in yeast and in vitro pull-down using the purified HP1gamma and Pim-1 expressed in Escherichia coli showed that Pim-1 directly bound to the chromo shadow domain of HP1gamma . HP1gamma was also associated with Pim-1 in human HeLa cells and the serine clusters located at the center of HP1gamma were phosphorylated by Pim-1 in vitro . Furthermore, a transcription repression activity of HP1gamma was further stimulated by the deletion of the serine clusters targeted by Pim-1 . These results suggest that Pim-1 affects the structure or silencing of chromatin by phosphorylating HP1.

Arch Virol, 1999, 144(12), 2449 - 56
Expression and characterization of the 3a movement protein of cowpea chlorotic mottle bromovirus; Fujita M et al.; Cowpea chlorotic mottle bromovirus (CCMV) 3a protein is required for cell-to-cell movement of the virus in host plants . The 3a protein was produced in Escherichia coli using an expression vector . Gel retardation analysis and UV cross-linking experiments demonstrated that the CCMV 3a protein (CC3a) bound single-stranded (ss) RNA cooperatively without sequence specificity . Binding competition analysis showed that CC3a bound ss-nucleic acids more strongly than double-stranded nucleic acids.

Immunogenetics, 2000 Jan, 51(1), 59 - 64
Molecular cloning, expression, and purification of pig interleukin-5; Sylvin H et al.; Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammation . Pig IL-5 was cloned, sequenced, and expressed to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced late-phase reactions . These pigs were sensitized to proteins extracted from Ascaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia . Peripheral blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulated . Total RNA was extracted and reverse transcribed into cDNA . IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymerase chain reaction product . 3' rapid amplification of cDNA ends (3'RACE) and 5'RACE procedures were applied to identify the 3' and 5' ends, respectively . The full-length pig IL-5 cDNA is 405 base pairs long . Mature pig IL-5 was expressed in Escherichia coli with a His-tag for purification . The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14 000 M(r) and forms a biologically active homodimer of 28 000 M(r) . Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep, horse, mouse, and rat counterparts.

J Exp Med, 2000 Feb 7, 191(3), 579 - 84
Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern; Wiesendanger M et al.; Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes . We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation . In Msh6(-)/- and Msh3(-)/-/Msh6(-)/- mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2(-)/- mice . In contrast, Msh3(-)/- mice show no differences from their littermate controls . These findings indicate that the MSH2-MSH6 heterodimer, but not the MSH2-MSH3 complex, is responsible for modulating Ig hypermutation.

J Exp Med, 2000 Feb 7, 191(3), 551 - 60
Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells; Alderson MR et al.; Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors . We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells . Using this technique, we identified a family of highly related Mtb antigens . The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A . Recombinant Mtb9.9A protein, expressed and purified from E . coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals . Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes . A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C . Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A . The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

Chem Biol, 2000 Jan, 7(1), 39 - 50
Multiple pathways of recombination define cellular responses to cisplatin; Zdraveski ZZ et al.; BACKGROUND: Cisplatin is a DNA-damaging drug used for treatment of testicular tumors . The toxicity of cisplatin probably results from its ability to form DNA adducts that inhibit polymerases . Blocked replication represents a particular challenge for tumor cells, which are committed to unremitting division . Recombination provides a mechanism by which replication can proceed despite the presence of lesions and therefore could be significant for managing cisplatin toxicity . RESULTS: Recombination-deficient Escherichia coli mutants were strikingly sensitive to cisplatin when compared with the parental strain . Our data identified both daughter-strand gap and double-strand break recombination pathways as critical for survival following treatment with cisplatin . Although it is established that nucleotide excision repair (NER) significantly protects against cisplatin toxicity, most recombination-deficient strains were as sensitive to the drug as the NER-deficient uvrA mutant . Recombination/NER deficient double mutants were more sensitive to cisplatin than the corresponding single mutants, suggesting that recombination and NER pathways play independent roles in countering cisplatin toxicity . Cisplatin was a potent recombinogen in comparison with the trans isomer and canonical alkylating agents . Mitomycin C, which like cisplatin, forms DNA cross-links, was also recombinogenic at minimally toxic doses . CONCLUSIONS: We have demonstrated that all of the major recombination pathways are critical for E . coli survival following treatment with cisplatin . Moreover, recombination pathways act independently of NER and are of equal importance to NER as genoprotective systems against cisplatin toxicity . Taken together, these results shed new light on how cells survive and succumb to this widely used anticancer drug.

Curr Biol, 2000 Jan 27, 10(2), 103 - 6
RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer; George H et al.; The Escherichia coli RuvA and RuvB proteins promote the branch migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in {1}) . Biochemical and structural studies of the RuvAB-Holliday junction complex have shown that RuvA binds directly to the Holliday junction {2} {3} {4} {5} {6} and acts as a specificity factor that promotes the targeting of RuvB {7} {8}, a hexameric ring protein that drives branch migration {9} {10} {11} . Electron microscopic visualisation of the RuvAB complex revealed that RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diametrically opposed across the junction {8} . ATP-dependent branch migration occurs as duplex DNA is pumped out through the centre of each ring . Because RuvB possesses well-conserved helicase motifs and RuvAB exhibits a 5'-3' DNA helicase activity in vitro {12}, the mechanism of branch migration is thought to involve DNA opening within the RuvB ring, which provides a single strand for the unidirectional translocation of the protein along DNA . We have investigated whether the RuvB ring can translocate along duplex DNA containing a site-directed interstrand psoralen crosslink . Surprisingly, we found that the crosslink failed to inhibit branch migration . We interpret these data as evidence against a base-by-base tracking model and suggest that extensive DNA opening within the RuvB ring is not required for DNA translocation by RuvB.

Virology, 2000 Feb 15, 267(2), 209 - 19
Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection; Guo J et al.; P56 is the most abundant protein induced by interferon (IFN) treatment of human cells . To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography . A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein . Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus . P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h . IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it . Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction . Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA . Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm .

Biotechnol Prog, 2000 Jan-Feb, 16(1), 86 - 91
Design of affinity tags for one-step protein purification from immobilized zinc columns; Pasquinelli RS et al.; Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography . Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins . Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins . For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0 . Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract . In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile . These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Biol Chem, 1999 Dec, 380(12), 1455 - 9
Molecular cloning and characterization of a cDNA encoding a transferrin homolog from Bombyx mori; Yun EY et al.; We isolated a cDNA representing a message that was strongly induced by injection with E . coli in Bombyx mori . The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa . The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding . We suggest that the B . mori transferrin plays an important role in the self-defense system.

Biol Chem, 1999 Dec, 380(12), 1405 - 11
Xylose utilisation: cloning and characterisation of the xylitol dehydrogenase from Galactocandida mastotermitis; Habenicht A et al.; We cloned and successfully expressed the gene for xylitol dehydrogenase from Galactocandida mastotermitis in Escherichia coli . The amino acid sequence revealed that the enzyme belongs to the superfamily of zinc containing, medium-chain alcohol dehydrogenases . The enzyme catalyses the second step in the xylose utilising pathway converting xylose to xylulosephosphate . Xylulose-phosphate is further degraded by the transaldolase and transketolase reactions of the pentose phosphate pathway . The purified xylitol dehydrogenase from G . mastotermitis was subjected to partial amino acid sequence analysis . The resulting amino acid information was then used to construct oligonucleotide probes for PCR amplification . The PCR product was used to screen a genomic library . The identified xdh gene includes one short intron at its 5' end . Putative regulatory signals were identified with the help of Saccharomyces cerevisiae regulatory sequence databases . An intronless xdh transcript, cloned by RT-PCR, was actively expressed in pBTac1 at 37 degrees C to approximately 8% of the soluble E . coli protein . Furthermore, the kinetic parameters were determined and conditions were found to stabilise the soluble and active protein.

Biol Chem, 1999 Dec, 380(12), 1395 - 403
Xylose utilisation: cloning and characterisation of the Xylose reductase from Candida tenuis; Hacker B et al.; Xylose reductases catalyse the initial reaction in the xylose utilisation pathway, the NAD(P)H+H+ dependent reduction of xylose to xylitol . In this work, the xylose reductase gene from Candida tenuis CBS 4435 was cloned and successfully expressed in E . coli . From the purified and partially sequenced protein primers were deduced for PCR . The fragment obtained was used for Southern blot analysis and screening of a subgenomic library . The clone containing the open reading frame was sequenced; the gene consisted of 969 nucleotides coding for a 322 amino acids protein with a molecular mass of 36 kDa . Putative regulatory signals were identified with the help of a Saccharomyces cerevisiae regulatory sequence database . In order to express the xylose reductase in E . coli, the gene was placed under positive and negative control . At low temperatures, the xylose reductase was expressed in soluble and active form up to about 10% of the soluble protein; with rising temperatures formation of visible inclusion bodies occurred . In refolding experiments we were able to recover the major portion of xylose reductase activity from the pellet fraction.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1999 Nov, 124(3), 309 - 14
Dietary L-arginine level alters plasma nitric oxide and apha-1 acid glycoprotein concentrations, and splenocyte proliferation in male broiler chickens following Escherichia coli lipopolysaccharide injection; Takahashi K et al.; Experiments were conducted to determine the effect of dietary arginine (Arg) on nitric oxide (NO) production following injection with Escherichia coli lipopolysaccharide (LPS) and splenocyte proliferative response to concanavalin A in broilers . Birds were fed experimental diets containing Arg at levels of 6.5, 14.4 or 24.4 g/kg diet in experiment 1, and 6.5, 14.4 or 19.3 g/kg diet in experiment 2, respectively, for 7 days and were then intraperitoneally injected with LPS (2 mg/kg body weight) after 16 h fasting . Maximum NO production estimated by plasma nitrite concentration was observed 6 or 10 h after LPS injection in all Arg groups . Dietary Arg level and/or intake were positively associated with NO production . NO production at 6 or 10 h after LPS injection coincided with changes in plasma alpha-1 acid glycoprotein concentration, an acute phase substance, at 10 or 24 h post-LPS injection . Splenocyte proliferation in chicks fed on Arg-sufficient (14.4 g/kg) diet was greater that that in chicks fed Arg-deficient or -excess diets . The results suggest that dietary Arg level and/or intake proportionally affect NO production, and acute phase inflammatory responses following LPS injection, and that marginal deficiency or excess of dietary Arg might reduce splenocyte proliferative responses.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1671 - 6
Suppression of chromosome segregation defects of Escherichia coli muk mutants by mutations in topoisomerase I; Sawitzke JA et al.; Escherichia coli muk mutants are temperature-sensitive and produce anucleate cells . A spontaneously occurring mutation was found in a DeltamukBkan mutant strain that suppressed the temperature-sensitive phenotype and mapped in or near topA, the gene that encodes topoisomerase I . Previously characterized topA mutations, topA10 and topA66, were found to be general suppressors of muk mutants: they suppressed temperature sensitivity and anucleate cell production of cells containing null or point mutations in mukB and null mutations in mukE or mukF . The suppression correlated with excess negative supercoiling by DNA gyrase, and the gyrase inhibitor, coumermycin, reversed it . Defects in topA allow 99% of cell division events in muk null mutants to proceed without chromosome loss or loss of cell viability . This observation imposes important limitations on models for Muk activity and is consistent with a role for MukBEF in chromosome folding and DNA condensation.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1554 - 9
Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?
Vos MH, Borisov VB, Liebl U, Martin JL, Konstantinov AA.
Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy . The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595) . The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d . In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d . Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps . This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein . Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

J Biol Chem, 2000 Feb 11, 275(6), 4513 - 8
Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids; Makise M et al.; DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be regulated through its binding to acidic phospholipids, such as cardiolipin . In our previous paper (Hase, M., Yoshimi, T., Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T . (1998) J . Biol . Chem . 273, 28651-28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg(360), Arg(364), and Lys(372)) were mutated to acidic amino acids showed a decreased ability to interact with cardiolipin in vitro, suggesting that DnaA protein binds to cardiolipin through an ionic interaction . In this study, we construct three mutant dnaA genes each with a single mutation and examined the function of the mutant proteins in vitro and in vivo . All mutant proteins maintained activities for DNA replication and ATP binding . A mutant protein in which Lys(372) was mutated to Glu showed the weakest interaction with cardiolipin among these three mutant proteins . Thus, Lys(372) seems to play an important role in the interaction between DnaA protein and acidic phospholipids . Plasmid complementation analyses revealed that all these mutant proteins, including DnaA431 could function as an initiator for chromosomal DNA replication in vivo.

J Biol Chem, 2000 Feb 11, 275(6), 4210 - 4
Low resolution structure of the sigma54 transcription factor revealed by X-ray solution scattering; Svergun DI et al.; The sigma54 RNA polymerase holoenzyme functions in enhancer-dependent transcription . The structural organization of the sigma54 subunit of bacterial RNA polymerase in solution is analyzed by synchrotron x-ray scattering . Scattering patterns are collected from the full-length protein and from a large fragment able to bind the core RNA polymerase, and their low resolution shapes are restored using two ab initio shape determination techniques . The sigma54 subunit is a highly elongated particle, and the core binding fragment can be unambiguously positioned inside the full-length protein . The boomerang-like shape of the core binding fragment is similar to that of the atomic model of a fragment of the Escherichia coli sigma70 protein, indicating that, although the sigma54 and sigma70 factors are unrelated by primary sequence, they may share some structural similarity . Potential DNA binding surfaces of sigma54 are also predicted by comparison with the sigma54 core binding fragment.

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