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 l