J Biol Chem, 1999 Mar 26, 274(13), 8764 - 9 Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity; Matkovic-Calogovic D et al.; Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined . The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus . Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75% . The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry . On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered . The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors . Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells. J Biol Chem, 1999 Mar 26, 274(13), 8723 - 9 Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy; Agrawal RK et al.; The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy . Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration {10-15 mM}; and polyamine buffer: containing Hepes, lower Mg2+ concentration {6 mM}, and polyamines) . Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome . The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions . The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i . e . in the P/P state, under the polyamine buffer conditions. J Biol Chem, 1999 Mar 26, 274(13), 8717 - 22 Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase; Shenoy SK et al.; The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3 . The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent . Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine . QPs3 was released from the fusion protein by proteolytic cleavage with thrombin . Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase . Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million . The recombinant QPs3 binds ubiquinone, causing a spectral blue shift . Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state . Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone . Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride . Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92 . Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride. J Biol Chem, 1999 Mar 26, 274(13), 8359 - 62 Caspase-9 can be activated without proteolytic processing; Stennicke HR et al.; The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coli is processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP . Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms . To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen . These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors . The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells . Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity. J Biol Chem, 1999 Mar 26, 274(13), 8351 - 4 A model of the transition state in the alkaline phosphatase reaction; Holtz KM et al.; A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 A resolution . The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile . A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site . This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzyme-catalyzed reaction. Infect Immun, 1999 Apr, 67(4), 2045 - 9 beta1-chain integrins are not essential for intimin-mediated host cell attachment and enteropathogenic Escherichia coli-induced actin condensation; Liu H et al.; Intimin is a bacterial outer membrane protein required for intimate attachment of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) to mammalian cells . beta1-chain integrins have been proposed as candidate receptors for intimin . We found that binding of mammalian cells to immobilized intimin was not detectable unless mammalian cells were preinfected with EPEC or EHEC . beta1-chain integrin antagonists or inactivation of the gene encoding the beta1-chain did not affect binding of preinfected mammalian cells to intimin or the actin condensation associated with the attachment of EPEC . The results indicate that beta1-chain integrins are not essential for intimin-mediated cell attachment or EPEC-mediated actin polymerization. Infect Immun, 1999 Apr, 67(4), 1821 - 7 Antibodies reactive with the N-terminal domain of Plasmodium falciparum serine repeat antigen inhibit cell proliferation by agglutinating merozoites and schizonts; Pang XL et al.; The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum . Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro . Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages . When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated . Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC) . Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation . These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release. Infect Immun, 1999 Apr, 67(4), 1633 - 9 Lipopolysaccharide enhances the production of vascular endothelial growth factor by human pulp cells in culture; Matsushita K et al.; We investigated whether vascular endothelial growth factor (VEGF) production by human pulp cells (HPC) is regulated by lipopolysaccharide (LPS) in relation to the pathogenesis of pulpitis . Although HPC incubated with medium alone only marginally expressed VEGF mRNA and produced a low level of VEGF as detected by enzyme-linked immunosorbent assay, the VEGF mRNA expression and VEGF production were markedly enhanced upon stimulation with LPS from Escherichia coli . Prevotella intermedia LPS, phorbol 12-myristate 13-acetate, and interleukin-6 also induced VEGF mRNA expression in HPC . A simian virus 40-infected HPC line also exhibited increased VEGF mRNA expression in response to E . coli LPS, but lung and skin fibroblasts did not . Fetal bovine serum (FBS) increased the sensitivity of HPC to LPS in a dose-dependent manner . HPC did not express membrane CD14 on their surfaces . However, the anti-CD14 monoclonal antibody MY4 inhibited VEGF induction upon stimulation with LPS in HPC cultures in the presence of 10% FBS but not in the absence of FBS . LPS augmented the VEGF production in HPC cultures in the presence of recombinant human soluble CD14 (sCD14) . To clarify the mechanisms of VEGF induction by LPS, we examined the possible activation of the transcription factor AP-1 in HPC stimulated with LPS, by a gel mobility shift assay . AP-1 activation in HPC was clearly observed, whereas that in skin fibroblasts was not . The AP-1 inhibitor curcumin strongly inhibited LPS-induced VEGF production in HPC cultures . In addition, a protein synthesis inhibitor, cycloheximide, inhibited VEGF mRNA accumulation in response to LPS . These results suggest that the enhanced production of VEGF in HPC induced by LPS takes place via an sCD14-dependent pathway which requires new protein synthesis and is mediated in part through AP-1 activation. Genet Anal, 1999 Feb, 14(5-6), 181 - 6 Enzymatic methods for mutation scanning; Taylor GR et al.; Enzymatic methods for mutation scanning still lack the sensitivity and specificity of the chemical cleavage of mismatch method . However developments in our understanding of the mismatch recognition process should lead to improvements . Several promising candidates exist with potential for more specific and sensitive mutation detection. Biochim Biophys Acta, 1999 Mar 19, 1430(2), 290 - 301 Systematic mutations of highly conserved His49 and carboxyl-terminal of recombinant porcine liver NADH-cytochrome b5 reductase solubilized domain; Kimura S et al.; The cDNA encoding solubilized porcine liver NADH-cytochrome b5 reductase catalytic domain (Pb5R) was cloned and overexpressed in Escherichia coli . A highly conserved His49 and a C-terminal Phe272 of Pb5R, which are located near the isoalloxazine moiety of the FAD, were systematically modulated by site-directed mutagenesis . Large structural change was not detected on the absorption and circular dichroism spectra of mutant proteins . Drastic changes in enzymatic properties were not observed, but the apparent Km value for soluble form of porcine liver cytochrome b5 (Pb5) was affected by the substitutions of His49 with glutamic acid and with lysine, deletion of C-terminal Phe272, and addition of Gly273 . The values of the catalytic constant (kcat) were obviously decreased by the substitution of His49 with glutamic acid or the addition of Gly273 . In these two mutants, the rate for reduction of FAD was decreased, and the rate for autoxidation of reduced FAD was increased . These results showed that His49 and C-terminal carboxyl group in Pb5R are not critical for the electron transfer to Pb5, but the electrostatic environmental changes at these positions could affect the recognition of Pb5 and modulate the catalytic function of the enzyme by changing the stability of reduced FAD. Biochim Biophys Acta, 1999 Mar 19, 1430(2), 191 - 202 Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5; Wibbenmeyer JA et al.; Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced . The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding . Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains . The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG . Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system. Biochim Biophys Acta, 1999 Feb 10, 1430(1), 119 - 26 Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions; D'Silva I et al.; Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions . DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively . In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions . PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM) . Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends . The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively . These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP . The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed. Biochim Biophys Acta, 1999 Feb 10, 1430(1), 95 - 102 Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli; Hsu NC et al.; P450c21 catalyzes an important step in steroid synthesis . Its deficiency leads to symptoms of steroid imbalance . To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli . The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification . Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells . One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity . The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal . The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra . The D380 mutant protein had 3-fold reduction in peak heights in both spectra . This reduced heme binding resulted in 3-fold lower enzymatic activity. Mutat Res, 1999 Mar 10, 425(1), 55 - 69 Use of log-linear analysis to construct explanatory models for TDBP- and AFB1-induced mutation spectra in lacI transgenic animals; Brackley ME et al.; Mutation spectra recovered from lacI transgenic animals exposed in separate experiments to tris-(2,3-dibromopropyl)phosphate (TDBP) or aflatoxin B1 (AFB1) were examined using log-linear analysis . Log-linear analysis is a categorical procedure that analyses contingency table data . Expected contingency table cell counts are estimated by maximum likelihood as effects of main variables and variable interactions . Evaluation of hierarchical models of decreasing complexity indicates when significant explanatory power is lost by the sequential omission of interactions between variables . Use of this technique allows construction of the most parsimonious models to account for mutation spectra obtained in the two experiments . The resulting statistical models are consistent with previous analyses of these data and with biological explanations for causes of the observed spectra . Mutat Res, 1999 Mar 10, 425(1), 47 - 54 The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques; Hill KA et al.; There are mutational artifacts in the Big Blue(R) assay and it is important to characterize the source and nature of these mutations . Differences were reported in the mutation patterns of a small sample of 23 sectored and 91 circular mutant plaques derived from skin using the Big Blue(R) transgenic mouse mutation detection system {G . R . Stuart, N.J . Gorelick, J.L . Andrews, J.G . de Boer, B.W . Glickman, The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice, Environ . Mol . Mutagen 28 (1996) 385-392.} . We have extended these observations by analyzing 46 sectored and 224 circular mutant plaques derived from seven tissues . The frequency of sectored mutant plaques is estimated to be 16% with no significant variation with tissue type . However, the patterns of mutation for sectored mutants and mouse-derived mutations differed significantly (p=0.04) . Base substitutions in sectored mutant plaques do not show the asymmetries found in circular mutants consistent with integration of a GC rich transgene into the AT rich mammalian genome . Sectored mutants have mutation patterns consistent with a mixture of mouse, in vitro and Escherichia coli-derived mutations . Data on the relative frequencies of different mutant plaque morphologies suggests that overlapped plaques are substantially contaminated by sectored plaques at recommended plating densities . Biochem Biophys Res Commun, 1999 Feb 5, 255(1), 123 - 8 Cloning and expression of two carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase cDNAs in ovarian follicles of rainbow trout (Oncorhynchus mykiss); Guan G et al.; In salmonid fish, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) is a key enzyme involved in the production of oocyte maturation-inducing hormone (MIH), 17alpha, 20beta-dihydroxy-4-pregnen-3-one . Here we report the isolation of two cDNAs which encode proteins with high homology to carbonyl reductase-like 20beta-HSD (CR/20beta-HSD) from rainbow trout (Oncorhynchus mykiss) ovarian follicles . Genomic DNA analysis showed that the two CR/20beta-HSD cDNAs are derived from two different genes . Northern blot and RT PCR analysis demonstrated that trout CR/20beta-HSDs are broadly expressed in various tissues . Enzymatic characterization using recombinant CR/20beta-HSD proteins produced in E . coli showed that the product of one of the two cDNAs had both 20beta-HSD and CR activity, but the other had neither activity . Although the functional significance of the two genes remains unresolved, these results clearly demonstrate the presence of two distinct CR/20beta-HSD transcripts in the trout ovary . Biochem Biophys Res Commun, 1999 Feb 5, 255(1), 1 - 5 R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase; Ohsato T et al.; Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells . The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase . A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA . Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication . This mitochondrial R-loop is resolved by RecG in a dose-dependent manner . Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG . A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop . This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase . The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG . Proteins, 1999 Mar 1, 34(4), 533 - 9 Characterization of internal motions of Escherichia coli ribonuclease H by Monte Carlo simulation; Haliloglu T; The backbone dynamics of Escherichia coli ribonuclease H (RNase H) is studied by a recently developed off-lattice Monte Carlo/Metropolis simulation technique . A low-resolution model (virtual-bond model) is used together with knowledge-based potentials . The calculated mean-square fluctuations in alpha carbons are in good agreement with crystallographic temperature factors . The conformations generated around the native state are analyzed by time-dependent orientational and conformational correlation functions to study the internal motions of RNase at different time windows . A correlation between the free-energy changes for native-state hydrogen exchange (HX) and the extent of the autocorrelation in the rotations of the virtual bonds at long times has been observed . Cross-correlations between the rotations of the bonds, which are near-neighbor in the sequence, are effective in all time windows and help the secondary structures to preserve their kinetic stability . On the other hand, the existence of cross-correlations at long times help the tertiary contacts be maintained . The order parameter of NH bond vector for each residue has been calculated and compared with 15N-NMR relaxation measurements. Mol Cell Biol, 1999 Apr, 19(4), 3177 - 83 Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations; Shcherbakova PV et al.; Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer . In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function . First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation . Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function . Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis . Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations . Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented . Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain . This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele. Virus Res, 1999 Feb, 59(2), 203 - 10 Immunological characterization of two major secreted forms of recombinant hepatitis B virus e antigens; Hwang GY et al.; Plasmids containing PCR-amplified hepatitis B virus e antigen (HBeAg) genes (HBeAg-MV and HBeAg-SV) were constructed and expressed in E . coli strain DH5alpha . The induced intracellular glutathione S-transferase (GST) fusion proteins of HBeAg-MV and HBeAg-SV were recovered and purified from bacterial lysates by affinity chromatography with glutathione-sepharose beads . The HBeAg-MV protein contained an additional 19 amino acids at its amino terminus . These two proteins were specifically cleaved from GST by the protease factor Xa and recognized by a monoclonal antibody against HBeAg . HBeAg-MV and HBeAg-SV were found to be the two major components of the post-modified HBcAg during viral infection . The antigenic specificities of the fusion and purified HBeAgs (factor Xa-digested) were confirmed by the Abbott HBe enzyme immunoassay (EIA) detection system . Sera from patients with confirmed hepatocellular carcinoma (HCC) specifically reacted only with HBeAg moiety of fusion proteins . HCC sera bound more strongly to the HBeAg-SV protein than to the HBeAg-MV one . This indicates that HBeAg-SV is either more antigenic than -MV or is the major target protein for the elicitation of antibody production after HBV infection . Thus, the two recombinant HBeAgs expressed and obtained in this study are appropriate immunological agents for the diagnostic detection of hepatitis B virus infection in humans. Virus Res, 1999 Feb, 59(2), 165 - 77 Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant; Osterrieder N; An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC) . The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene . On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC) . However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus . Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations . These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell . Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used . L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells . Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia . Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence. Protein Sci, 1998 Aug, 7(8), 1839 - 42 Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis; Kinsland C et al.; A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described . This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively . The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed. Protein Sci, 1998 Aug, 7(8), 1836 - 8 Crystallization of recombinant human heme oxygenase-1; Schuller DJ et al.; Heme oxygenase catalyzes the NADPH, O2, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide . One of two primary isozymes, HO-1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C-terminus . While full-length human HO-1 consists of 288 residues, a truncated version with residues 1-265 has been expressed as a soluble active enzyme in Escherichia coli . The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies . SDS gel analysis indicated that the protein had undergone proteolytic degradation . An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize . N-terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1-226 and 1-237 . Expression of the 1-226 and 1-233 versions of human HO-1 provided active enzyme that crystallizes in a form suitable for diffraction studies . These crystals belong to space group P2(1), with unit cell dimensions a = 79.3 A, b = 56.3 A, c = 112.8 A, and beta = 101.5 degrees. Protein Sci, 1998 Aug, 7(8), 1821 - 8 Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein; Kim K et al.; We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein . IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds . A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets . Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically . These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse . We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997) . Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions . For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants . In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step . Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding. Protein Sci, 1998 Aug, 7(8), 1796 - 801 Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry; Kelleher NL et al.; The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy . Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli . Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences . For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses . Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue . For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide . The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated . For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein . MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences. Protein Sci, 1998 Aug, 7(8), 1781 - 8 The role of the 6 lysines and the terminal amine of Escherichia coli single-strand binding protein in its binding of single-stranded DNA; Chen J et al.; Differential chemical modification of the lysines and amino-terminus of Escherichia coli single-strand binding (SSB) protein was used to determine their roles in the binding of SSB to single-stranded DNA (ssDNA) . A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride . First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single-stranded ssDNA . Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride . Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry . The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA . Acetylation rate constants were determined from time course measurements . In the absence of ssDNA, the terminal alpha-amine of SSB was 10-fold more reactive than Lys residues at positions 43, 62, 73, and 87 . The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition . In the presence of ssDNA, the reactivities of the amino-terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7-25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA . Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer-monomer interface. Protein Sci, 1998 Aug, 7(8), 1757 - 67 Probing enzyme quaternary structure by combinatorial mutagenesis and selection; MacBeath G et al.; Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants . We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme . By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity . The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure . The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins. Protein Sci, 1998 Aug, 7(8), 1728 - 37 Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator; Vindigni A et al.; The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media . The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1) . Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin . In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2 . A specific inhibition of tPA by Cu2+ was discovered . The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM . In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases . The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI . Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme . Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E . coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr . These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases. J Vet Med Sci, 1999 Feb, 61(2), 171 - 3 Expression of bovine cytokines in Escherichia coli; Kashima T et al.; Glutathione S-transferase fusion proteins of bovine interleukin-2 (IL-2), IL-4, IL-6 and interferon-gamma (IFN-gamma) were expressed in Escherichia coli . Complementary DNA (cDNA) for open reading frame of each cytokine without signal peptide encoding region was amplified by reverse transcriptional polymerase chain reaction method and was subcloned into pGEX-5X-1 . In result, IL-6 and IFN-gamma fusion proteins in bacteria were soluble, but IL-2 and IL-4 fusion proteins were insoluble . The insoluble IL-2 fusion protein successfully refolded by urea became soluble . The recombinant IL-2, IL-6 and IFN-gamma could be obtained by the batch method using Glutathione Sepharose 4B and Factor Xa digestion, which may be useful for preparation of antisera as antigens and functional studies. Domest Anim Endocrinol, 1999 Jan, 16(1), 69 - 80 Effect of exogenous FSH on ovulation rate in homozygous carriers or noncarriers of the Booroola FecB gene after hypothalamic-pituitary disconnection or after treatment with a GnRH agonist; Hudson NL et al.; We have tested the hypothesis "that the ovulation rate in homozygous carriers (BB) and noncarriers (+2) of the Booroola FecB gene would not be different if the plasma concentrations of follicle-stimulating hormone (FSH) in the two genotypes were similar." For this purpose we used two experimental animal models: 1) the hypothalamic-pituitary disconnected (HPD) ovary-intact ewe; and 2) and GnRH agonist (i.e., Deslorelin)-treated ewe . Following HPD or Deslorelin treatment, the animals had low plasma concentrations of gonadotropins and were anovulatory . In both animal models, BB and +2 ewes were treated with exogenous pregnant mares serum gonadotropin (PMSG) and varying doses of FSH to induce preovulatory follicular growth, and human chorionic gonadotropin (hCG) to induce ovulation . HPD or Deslorelin-treated animals administered with pregnant mares serum gonadotropin without FSH followed by human chorionic gonadotropin failed to ovulate . However for both animal models, the proportion of BB and +2 ewes ovulating to various doses of FSH differed such that significantly greater proportions of +2 animals ovulated relative to the BB genotype (P < 0.05) . When HPD or Deslorelin-treated BB and +2 ewes were administered identical doses of FSH, the mean ovulation rate and plasma concentrations of FSH in those animals which ovulated was the same in both genotypes . These findings confirm, at least in part, the aforementioned hypothesis . The results also demonstrated that higher ovulation rates were obtained in both genotypes as the FSH dose was increased . Collectively, these findings infer that the higher mean ovulation rate in normal intact BB ewes compared to the +2 genotype is attributable to effects of the FecB gene at the level of ovarian follicular development as well as at the level of pituitary FSH release. Gesundheitswesen, 1999 Jan, 61(1), 38 - 44 {Environmental studies of asymptomatic kindergarten children as carriers of enterohemorrhagic Escherichia coli (EHEC) in the Ammerland district}; Vogelsang E et al.; The increase of serious EHEC diseases in the northwest Lower Saxony in summer 1997 was accompanied by a lively discussion on the hazard represented by this pathogen which gained a large amount of media attention . The Lower Saxony Public Health Department initiated a study of this case in the Weser-Ems government district to investigate the spread of EHEC by children and their supervisors in kindergartens receiving certified raw milk supply . This established that there were ten kindergarten children in the Ammerland rural district who were asymptomatik EHEC carriers . The Local Public Health Department immediately carried out the necessary epidemic hygiene measures . First of all, control investigations were performed on the affected children, including environmental investigations on their families and contracts . The results of these investigations revealed the need to carry out additional environmental investigations in two kindegartens (follow-up investigations) as well as amongst employees in a hospital canteen . Within the families, a mother of an affected child was also identified as another asymptomatic EHEC carrier . However, the strains that were isolated reflected different serotypes . In total, the investigation of 337 people in contact with the eleven asymptomatic EHEC carriers did not confirm any person-to-person transmission--even though three of the kindergarten children shedding the organisms for 6-10 weeks attended the kindergartens for at least 4 weeks . No EHEC disease occurred in the communal facilities, neither were any positive cases identified by additional control investigations . The results indicate a probable lower rate of infectiousness by healthy EHEC carriers than was previously thought to be the case . Further studies are needed to decide if in future one should proceed on a case by case basis when considering the reauthorization of affected children and other people to enter communal facilities. Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 532 - 6 Recognition of sequence-directed structure of the ssDNA backbone by nucleases; Akaboshi E; Escherichia coli endonuclease I and exonuclease VII appear to recognize sequence-dependent conformations in the ssDNA backbone . ssDNAs, containing either A- and/or T-tract or a CAP binding region, were digested with these nucleases under conditions which minimize the formation of secondary structures . The digestion patterns were examined in relation to previous results of biochemical and crystallographic studies on dsDNA, and showed broad agreement . Endonuclease I cleaved ssDNA at sites corresponding to bent sites in dsDNA . Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 500 - 4 Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis; Timofeevski SL et al.; Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium . Despite structural similarity, these peroxidases oxidize different substrates . Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+ . By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli . The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3 . The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes . The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3 . Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation . The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis . J Mol Biol, 1999 Mar 26, 287(2), 383 - 94 The crystal structure of Escherichia coli class II fructose-1, 6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity; Hall DR et al.; The structure of a class II fructose-1,6-bisphosphate aldolase in complex with the substrate analogue and inhibitor phosphoglycolohydroxamate (PGH) has been determined using X-ray diffraction terms to a resolution of 2.0 A (1 A=0.1 nm) . The crystals are trigonal, space group P3121 with a=b=78.24 A, c=289.69 A . The asymmetric unit is a homodimer of (alpha/beta)8 barrels and the model has refined to give R-work 19.2 %, R-free (based on 5 % of the data) 23.0 % . PGH resembles the ene-diolate transition state of the physiological substrate dihydroxyacetone phosphate . It is well ordered and bound in a deep polar cavity at the C-terminal end of the (alpha/beta)8 barrel, where it chelates the catalytic zinc ion using hydroxyl and enolate oxygen atoms . Trigonal bipyramidal coordination of the zinc ion is completed by three histidine residues . The complex network of hydrogen bonds at the catalytic centre is required to organise the position of key functional groups and metal ion ligands . A well-defined monovalent cation-binding site is observed following significant re-organisation of loop structures . This assists the formation of a phosphate-binding site on one side of the barrel that tethers PGH in the catalytic site . The positions of functional groups of substrate and putative interactions with key amino acid residues are identified . Knowledge of the complex structure complements the results of spectroscopic and site-directed mutagenesis studies, and contributes to our understanding of the mechanism and substrate specificity of this family of enzymes . A reaction mechanism distinct from that proposed for other class II aldolases is discussed . The results suggest that the class II aldolases should be sub-divided into two groups on the basis of both distinct folds and mechanism . J Mol Biol, 1999 Mar 26, 287(2), 331 - 46 Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase; Putnam CD et al.; Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi) . A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity . High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi . The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes . Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA . Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex . Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands . A similar beta-zipper is observed in the conversion between the open and closed forms of UDG . The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity . J Mol Biol, 1999 Mar 26, 287(2), 277 - 85 A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila; Simmons AJ et al.; In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis . Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus . We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e . CPS.B2 domain) of CPSase . This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases . When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype . Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition . The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP . However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values . From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site . This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters . J Mol Biol, 1999 Mar 26, 287(2), 265 - 76 Evolution of differential substrate specificities in Mu class glutathione transferases probed by DNA shuffling; Hansson LO et al.; A library of variant enzymes was created by combined shuffling of the DNA encoding the human Mu class glutathione transferases GST M1-1 and GST M2-2 . The parental GSTs are 84 % sequence identical at the protein level, but their specific activities with the substrates aminochrome and 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG) differ by more than 100-fold . Aminochrome is of particular interest as an oxidation product of dopamine and of possible significance in the etiology of Parkinson's disease, and cyanoDMNG is a model for genotoxic and potentially carcinogenic nitroso compounds . GST M2-2 has at least two orders of magnitude higher catalytic activity with both of the substrates than any of the other known GSTs, including GST M1-1 . The DNA library of variant Mu class GST sequences contained "mosaic" structures composed of alternating segments of both parental sequences . All clones contained the 5'-end of a GST M1-1 clone optimized for high-level expression in Escherichia coli . The remainder of the sequences derived from segments of GST M2-2 and GST M1-1 DNA . All of the clones analyzed contained between two and seven distinct DNA segments . In addition, each clone contained an average of approximately one point mutation . None of the library clones analyzed was identical with either of the two parental structures . Variant GST sequences were expressed in E . coli, and their enzymatic activities with aminochrome, cyanoDMNG, and 1-chloro-2,4-dinitrobenzene (CDNB) were determined in bacterial lysates . Such screening of more than 70 clones demonstrated a continuous range of activities covering at least two orders of magnitude for each of the substrates . For a given clone, the activities with aminochrome and cyanoDMNG, in spite of their different chemistries, were clearly correlated, whereas no strong correlation was found with CDNB . This functional correlation suggests a common structural basis for the enzymatic mechanisms for conjugation of aminochrome and denitrosation of cyanoDMNG . From an evolutionary perspective, the results show that recombination of segments from homologous proteins gives rise to a large proportion of functionally competent proteins with a range of activities . The data support the proposal that natural evolution of protein functions may involve recombination of DNA segments followed by selection for advantageous functional properties of the resulting proteins . Clearly, the same approach can be utilized in the engineering of proteins displaying novel functions by in vitro evolution . Plant Mol Biol, 1999 Jan, 39(2), 381 - 6 Random mutagenesis in the large extrinsic loop E and transmembrane alpha-helix VI of the CP 47 protein of Photosystem II; Wu J et al.; The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution . Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane alpha-helix VI . Four mutant cell lines were recovered in which the histidyl residues 455H, 466H and 469H were altered . The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455H, 466H and 469H as chlorophyll ligands . Four additional mutant cell lines were recovered which contained mutations at positions 448R in the large extrinsic loop of CP 47 . These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters . Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations . Finally, a mutant cell line exhibiting a substitution at position 342G was recovered . The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution . In addition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced . These mutants uniformly lacked the ability to either grow photoautotrophically or evolve oxygen. Mol Biochem Parasitol, 1999 Jan 25, 98(2), 225 - 37 Molecular characterization of a calponin-like protein from Schistosoma japonicum; Yang W et al.; The gene for a Schistosoma japonicum (Philippine strain origin) (Sjp) calponin-like protein has been cloned and characterised . The clone, designated P14, was isolated from a Sjp adult worm lambda ZAP cDNA library by immunoscreening, and was shown to contain a full-length cDNA encoding a 38.3 kDa protein that shared significant sequence similarity to a number of previously reported calponins and 22 kDa smooth-muscle proteins . Northern analysis indicated the P14 transcript was approximately 2.2 kb in both Sjp and Chinese strain S . japonicum (Sjc) adult worms . Southern blot analysis of genomic DNA suggested that several copies of the P14 gene are present in the Sjc and Sjp genomes but only one copy was evident in the S . mansoni (Sm) genome . Western blot analysis indicated that the product of P14 occurs as a 38 kDa protein in adult Sjp worms and homologues are present in adult worms of Sjc and Sm . At least six isoforms, all with a similar molecular size of approximately 38 kDa and isoelectric points ranging from 8.1 to 9.5, were present in adult Sjc worms . The protein was immunolocalized to the muscle of male and female Sjc adult worms . Recombinant protein was expressed in E . coli and purified under denaturing conditions, and in yeast to produce a soluble protein in purified form . The availability of purified, correctly folded protein will allow investigations into its biological functions and potential involvement in host immunity. Bioorg Khim, 1998 Dec, 24(12), 920 - 5 {A new inhibitor of pyrimidine phosphorylase}; Dmitrieva NA et al.; 5,5-Bis(hydroxymethyl)-2-oxo-{1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases . Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly . Its LD50 in mice was 40 mg/kg . Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy. FEMS Microbiol Lett, 1999 Mar 1, 172(1), 91 - 7 Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids; Chao KL et al.; The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events . Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell . The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids . These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity. FEMS Microbiol Lett, 1999 Mar 1, 172(1), 29 - 34 An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli; Lai XH et al.; Representative strains of the different diarrheagenic Escherichia coli virotypes were tested for their potential cytotoxicity in the J774 macrophage cell line . All the seven virotypes of E . coli were cytotoxic to J774 macrophages, and in most cases the bacteria induced an apoptotic response . With the exception of the enterotoxigenic E . coli (ETEC) strain, all the other six virotypes caused induction of apoptosis as evidenced by quantitative analysis of the characteristic DNA fragmentation at the individual cell level . These results suggest that apoptosis could be one of the mechanisms contributing to the diarrheal disease development. Dev Genes Evol, 1999 Mar, 209(3), 145 - 54 Segmentation gene expression in the mothmidge Clogmia albipunctata (Diptera, psychodidae) and other primitive dipterans; Rohr KB et al.; To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, we studied the expression of two gap genes hunchback (hb) and Kruppel (Kr) as well as the pair-rule gene even-skipped (eve) in the dipteran Clogmia albipunctata (Nematocera, Psychodidae) . This species has features of both short- and long-germ mode of embryogenesis . In Clogmia hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region . These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis . Kruppel expression, on the other hand, was found to be rather similar to the Drosophila expression, both at early and late stages . eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension . Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans . We therefore also studied another nematoceran, Coboldia, to address this question and found that some segmental stripes form by intercalation as in Drosophila, although belatedly . Our results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects. Nature, 1999 Mar 4, 398(6722), 84 - 90 Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase; Meng W et al.; Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface . The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity . Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential . In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70 . Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70 . The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein . The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation . In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket . Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module. Nature, 1999 Mar 4, 398(6722), 39 - 46 Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport; Vetter IR et al.; The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport . The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes . The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex . The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end . This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP . Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein. J Mol Recognit, 1998 Winter, 11(1-6), 91 - 3 Phage viability in organic media: insights into phage stability; Olofsson L et al.; The stability of the filamentous phages derived from phagemid pG8H6 has been examined in a range of solvents and solvent mixtures . The results show an enhanced capacity to infect E . coli after exposure to various organic solvent-water mixtures . The dependence of stability upon solvent hydrophobicity was demonstrated . Furthermore, conditions have been identified which should allow the application of phage display libraries based upon pG8H6 in organic media. Biochemistry, 1999 Mar 16, 38(11), 3421 - 5 Curvature of dinucleotide poised for formation of trinucleotide in transcription with Escherichia coli RNA polymerase; Garland CS et al.; A frequently used schematic model of transcriptional elongation shows an RNA polymerase molecule moving along a linear DNA . This model is of course highly idealized and not compatible with promoter sequences {Gralla, J . D . (1991) Cell 66, 415-418; Schleif, R . (1992) Annu . Rev . Biochem . 61, 199-223} and regulatory proteins {Koleske, A . J., and Young, R . A . (1995) Trends Biochem . Sci . 20, 113-116; Dunaway, M., and Droge, P . (1989) Nature 341, 657-659; Muller, H . P., Sogo, J . M., and Schaffner, W . (1989) Cell 58, 767-777} located some distance away from the point of transcription initiation {Karsten, R., von Hippel, P . H., and Langowski, J . (1995) Trends Biochem . Sci . 20, 500-506} . These circumstances lead to the expectation of curvature along the DNA strand and require looping between sometimes distant points . We have now shown curvature in a dinucleotide formed at the very onset of transcription when it is poised for reaction with a mononucleotide to form a trinucleotide . The curvature became evident from the demonstration that a metal ion bound with a mononucleotide in the i+1 (elongation) site is approximately equidistant from bases at the 5' end (i-1 site) and 3' end (i site) of the dinucleotide . Similar results were obtained with three different dinucleotides and four mononucleotides . Curvature of the RNA initiate may reflect curvature of the DNA to which it is bound . These studies show curvature to be a significant feature in the interaction between DNA template and RNA elongate even at the very beginning of transcription. Biochemistry, 1999 Mar 16, 38(11), 3393 - 400 Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin; Blanchard CZ et al.; Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids . The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein . In this report a system for site-directed mutagenesis of the biotin carboxylase component is described . The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid . Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography . This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase . In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function . The mutations did not affect the bicarbonate-dependent ATPase reaction . In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin . The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations . The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes . The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme . Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase. Biochemistry, 1999 Mar 16, 38(11), 3335 - 44 Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects; D'Ham C et al.; Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg) . Three techniques of analysis were employed . A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage . Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT . This confirms that Tg is an excellent substrate for endonuclease III . Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III . Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine . While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS . From these data it may be suggested that 5-OHC could be modified during its enzymatic excision . Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected. Biochemistry, 1999 Mar 16, 38(11), 3327 - 34 Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site; Lundegaard C et al.; Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) . These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides . We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate . To investigate the role of this unusual proline (Pro 131 in the E . coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein . We found that UPRTase of E . coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil . The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays . The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil . The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected . We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate. Biochemistry, 1999 Mar 16, 38(11), 3280 - 4 Familial prion disease mutation alters the secondary structure of recombinant mouse prion protein: implications for the mechanism of prion formation; Cappai R et al.; A considerable body of data supports the model that the infectious agent (called a prion) which causes the transmissible spongiform encephalopathies is a replicating polypeptide devoid of nucleic acid . Prions are believed to propagate by changing the conformation of the normal cellular prion protein (PrPc) into an infectious isoform without altering the primary sequence . Proteins equivalent to the mature form of the wild-type mouse prion protein (residues 23-231) or with a mutation equivalent to that associated with Gerstmann-Straussler-Scheinker disease (proline to leucine at codon 102 in human; 101 in mouse) were expressed in E . coli . The mutation did not alter the relative proteinase K susceptibility properties of the mouse prion proteins . The wild-type and mutant proteins were analyzed by circular dichroism under different pH and temperature conditions . The mutation was associated with a decrease in alpha-helical content, while the beta-sheet content of the two proteins was unchanged . This suggests the mutation, while altering the secondary structure of PrP, is not sufficient to induce proteinase K resistance and could therefore represent an intermediate isoform along the pathway toward prion formation. Eur J Pharmacol, 1999 Feb 19, 367(2-3), 351 - 9 Central effects of cromoglycate sodium salt in rats treated with lipopolysaccharide; Nava F et al.; In 24-h water- and food-deprived rats, we have evaluated the effects of cromoglycate sodium salt, an inhibitor of the mast cell degranulation with anti-inflammatory and membrane-stabilizating activity, on the central effects induced by Escherichia coli lipopolysaccharide (LPS) . Intraperitoneal (i.p.) injection of LPS (0.25, 0.50 and 1 mg/kg) induced a dose-dependent inhibition of water and food intake, fever, reduction in locomotor activity as well as increased anxiety levels . All these LPS effects were antagonized by a prior intracerebroventricular (i.c.v.) injection of cromoglycate sodium salt (100, 150 and 200 microg/rat) . Our findings suggest that peripheral LPS administration may activate brain mast cells and indicate an involvement of these cells in brain pathophysiology. Bull Soc Pathol Exot, 1998, 91(5 Pt 1-2), 450 - 1 {Traveller's diarrhea: progress and lessons drawn from recent surveys}; Steffen R; Diarrhea among travellers continues to be as widespread as ever . A multicentric investigation carried out in Jamaica, Kenya, Goa (India) and Fortaleza (Brazil) indicates high incidence rates and enterotoxigenic Escherichia coli are the most common etiology for each destination. Mol Cell, 1999 Feb, 3(2), 255 - 61 hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA; Gradia S et al.; Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch . Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular . We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone . Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches. Mol Cell, 1999 Feb, 3(2), 239 - 45 ISWI is an ATP-dependent nucleosome remodeling factor; Corona DF et al.; The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin . We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions . The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes . Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes . The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs. FEMS Microbiol Lett, 1999 Feb 15, 171(2), 141 - 6 Increased heavy metal sensitivity of Escherichia coli producing the expression product of priA gene derived from the basidiomycete Lentinus edodes; Ishizaki T et al.; We have previously isolated a developmentally regulated novel gene, priA, from the basidiomycete Lentinus edodes . The deduced PRIA protein contains the two set of motifs similar to a 'zinc finger' typified by transcription factor TFIIIA and the motif of a 'zinc cluster' observed in metallothioneins . It also contains a hydrophobic N-terminal sequence . Here Escherichia coli cells producing PRIA were found to show a remarkable sensitivity to zinc ion and other heavy metal ions such as nickel and cadmium . Deletion analysis of PRIA revealed that the zinc-binding motifs and the hydrophobic N-terminal sequence are responsible for conferring the heavy metal sensitivity on the host cells. Ital J Gastroenterol Hepatol, 1998 Oct, 30 Suppl 3, S261 - 3 Experimental model of Helicobacter pylori infection; Del Giudice G et al.; Critical issues in the development of a vaccine against Helicobacter pylori are represented by the definition of molecules important in the pathogenesis of the infection, by the availability of an animal model reproducing several aspects of the human infection, and lastly by the availability of powerful adjuvants allowing strong protection after mucosal delivery of the antigens . A mouse model of Helicobacter pylori infection was established in our laboratories . Vaccination of these animals with Helicobacter pylori antigens, such as VacA, CagA, etc., induced protection, both prophylactic and therapeutic, when antigens were administered orally together with fully non toxic mutants of Escherichia coli heat-labile enterotoxin, as mucosal adjuvants . This experimental mouse model allows the study of the pathogenesis of Helicobacter pylori infection and the development of vaccines. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 3211 - 6 Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus; Wilson SP et al.; To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter . This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice . After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression . Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses . Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection . Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission . This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord . The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene . Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2964 - 9 Microsatellite instability in Drosophila spellchecker1 (MutS homolog) mutants; Flores C et al.; We have cloned a mutS homolog from Drosophila melanogaster called spellchecker1 (spel1) and have constructed spel1 mutant flies . MutS proteins promote the correction of DNA mismatches and serve important roles in DNA replication, recombination, and repair . The spel1 gene belongs to a subfamily of mutS first characterized by the MSH2 gene of yeast and which also includes hMSH2, one of the two major hereditary nonpolyposis colon cancer loci of humans . Like msh2 mutants in other species, we find that flies lacking the spel1 gene suffer a highly increased rate of instability in long runs of dinucleotide repeats when analyzed after 10-12 fly generations . Using a new assay, we have also discovered that mutations in spel1 decrease the stability of a dinucleotide repeat when it is copied into the site of a double-strand break during gene conversion . Contrary to the case in mammalian cells, spel1 deficiency does not affect tolerance of flies to a methylating agent nor does it affect resistance to gamma-irradiation. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2752 - 7 Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors; Sata M et al.; Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release . ARFs are approximately 20-kDa GTPases that are active with bound GTP and inactive with GDP bound . Conversion of ARF-GDP to ARF-GTP is regulated by guanine nucleotide-exchange proteins . All known ARF guanine nucleotide-exchange proteins contain a Sec7 domain of approximately 200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA) . To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7) . Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965-Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity . A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d) . Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect . As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2692 - 7 Identification of a transcription factor, encoded by two vaccinia virus early genes, that regulates the intermediate stage of viral gene expression; Sanz P et al.; Vaccinia virus early, intermediate, and late stage genes are sequentially transcribed by the viral RNA polymerase within the cytoplasm of infected cells . We found that the 34- and 45-kDa polypeptides encoded by vaccinia virus ORFs A8R and A23R, respectively, were necessary to reconstitute transcription of a template with an intermediate stage promoter . Coexpression of the A8R and A23R genes in Escherichia coli was required for in vitro activity . In addition, the two polypeptides copurified, indicating their association as protein subunits of a vaccinia virus intermediate transcription factor . This factor, which we named VITF-3, complemented three viral proteins-namely, the RNA polymerase, capping enzyme, and a 30-kDa protein called VITF-1 that is also a subunit of the RNA polymerase-and an unidentified cell factor called VITF-2 . Expression of the A8R and A23R genes occurred between 1 and 5 h after vaccinia virus infection and was not prevented by an inhibitor of DNA replication, consistent with a role for VITF-3 in specifically regulating intermediate transcription in vivo . The vaccinia virus A8R and A23R genes are highly conserved among vertebrate poxviruses, but no other viral or cellular homologs were identified. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2682 - 6 GroES in the asymmetric GroEL14-GroES7 complex exchanges via an associative mechanism; Horowitz PM et al.; The interaction of the chaperonin GroEL14 with its cochaperonin GroES7 is dynamic, involving stable, asymmetric 1:1 complexes (GroES7.GroEL7-GroEL7) and transient, metastable symmetric 2:1 complexes {GroES7.GroEL7-GroEL7.GroES7} . The transient formation of a 2:1 complex permits exchange of free GroES7 for GroES7 bound in the stable 1:1 complex . Electrophoresis in the presence of ADP was used to resolve free GroEL14 from the GroES7-GroEL14 complex . Titration of GroEL14 with radiolabeled GroES7 to molar ratios of 32:1 demonstrated a 1:1 limiting stoichiometry in a stable complex . No stable 2:1 complex was detected . Preincubation of the asymmetric GroES7.GroEL7-GroEL7 complex with excess unlabeled GroES7 in the presence of ADP demonstrated GroES7 exchange . The rates of GroES7 exchange were proportional to the concentration of unlabeled free GroES7 . This concentration dependence points to an associative mechanism in which exchange of GroES7 occurs by way of a transient 2:1 complex and excludes a dissociative mechanism in which exchange occurs by way of free GroEL14 . Exchange of radiolabeled ADP from 1:1 complexes was much slower than the exchange of GroES7 . In agreement with recent structural studies, this indicates that conformational changes in GroEL14 following the dissociation of GroES7 must precede ADP release . These results explain how the GroEL14 cavity can become reversibly accessible to proteins under in vivo conditions that favor 2:1 complexes. Bioconjug Chem, 1999 Mar-Apr, 10(2), 155 - 8 Paramagnetic oligonucleotides: contrast agents for magnetic resonance imaging with proton relaxation enhancement effects; Hines JV et al.; An antisense paramagnetic oligonucleotide analogue targeted to a model macromolecular receptor (5S rRNA) was prepared . The paramagnetic agent's relaxivity (dependence of the relaxation rate on paramagnetic agent concentration) in the presence and absence of the macromolecular receptor was measured at 1.5 and 6.3 T . The relaxivity of the targeted agent increased specifically in the presence of the macromolecular receptor (16% at 6.3 T and 15% at 1.5 T) . This effect was specific for a paramagnetic oligonucleotide targeted to the receptor and was larger than the relaxivity enhancement due simply to receptor-induced viscosity differences . Maximizing this relaxivity enhancement of tumor targeted paramagnetic oligonucleotides will aid in contrast agent development for magnetic resonance imaging. Int J Biol Macromol, 1999 Jan, 24(1), 65 - 7 Size and folding in globular proteins; Yan BC et al.; We have modeled protein folding by packing a unified length of regular structural elements (alpha-helices and beta-sheets) into a 'cube' . In a globular protein with m alpha-helices and n beta-strands, this unified length is expressed in units of heptapeptides in alpha-helices, and in units of tripeptides in beta-strands . Calculations using published data show that a 4-helix bundle (m = 4, n = 0) has at least 2 x 2 x 2 helical heptapeptides; the 16-strand beta-barrel of porin (m = 0, n = 16) is at most 4 x 4 x 4 tripeptides in beta-strands . Compact, recurring protein modules with mixed helices and beta-strands are the ones that actually acquire a geometrically quasi-spherical, or cubic, shape. Int J Biol Macromol, 1999 Jan, 24(1), 11 - 4 Structural and functional heterogeneity of the amino-terminal receptor-binding domain of human interferon-alpha 2; Kontsekova E et al.; Structural immunoanalysis of human interferon (IFN)-alpha 2c revealed antigenic and functional heterogeneity in its N-terminal receptor-binding domain (loop AB) . Monoclonal antibodies (mAbs) mapped to the region 30-53 of IFN-alpha 2 defined three partially overlapping antigenic sites designated here as 'a', 'b' and 'c' . For the high-affinity binding of IFN-alpha 2c to the cellular receptor, site b located in segment 34-41 and site c (residues 43-53) appeared to be most important . Only the part of site a (amino acids 30-33) seemed to be involved in the interaction with receptor . The segment of residues 30-46 forms a relatively straight structure on the protein surface, according to the three-dimensional model of human IFN-alpha 2. FEMS Immunol Med Microbiol, 1999 Feb, 23(2), 125 - 33 Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases; Takasuka T et al.; The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis . Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified . An A . fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe . Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli . Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified . These results indicate that A . fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB . The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A . fumigatus . Staining was observed heterogeneously throughout the fungal hyphae . This result indicates that catalase is produced by A . fumigatus during invasive aspergillosis. Surgery, 1999 Mar, 125(3), 339 - 44 Effects of endotoxin on regulation of intestinal smooth muscle nitric oxide synthase and intestinal transit; Cullen JJ et al.; BACKGROUND: The disrupted intestinal transit during endotoxemia may be mediated by nitric oxide (NO) . We hypothesized that the isoforms of nitric oxide synthase (NOS) are up-regulated in intestinal smooth muscle during endotoxemia and that the scavenging of NO will normalize transit . METHODS: Rats were given Escherichia coli lipopolysaccharide (LPS) 10 mg/kg intravenously and were killed 4 hours later . To determine the activity of NOS isoforms in the jejunum and ileum, the conversion of tritiated L-arginine to tritiated L-citrulline was measured . Western immunoblots were performed by incubating the extracted protein with specific polyclonal antibodies . To determine intestinal transit, rats were divided into 4 groups: 0.9% sodium chloride 1 mL/h intravenously for 5 hours, LPS 10 mg/kg intravenous bolus plus 1 mL/h 0.9% sodium chloride intravenously, LPS plus oxyhemoglobin 0.5 g/kg/h intravenously, and oxyhemoglobin 0.5 g/kg/h intravenously . RESULTS: LPS increased the constitutive and inducible NOS enzyme activities in the jejunum and ileum . Western blots demonstrated that LPS up-regulates both the NOS1 and NOS2 isoforms in jejunal and ileal smooth muscle . Oxyhemoglobin alone increased intestinal transit compared with controls, whereas endotoxemia increased intestinal transit, which was ameliorated with infusions of oxyhemoglobin . CONCLUSIONS: NO may play a major role in mediating the rapid intestinal transit induced by endotoxemia. Surgery, 1999 Mar, 125(3), 280 - 7 C1-esterase inhibitor and its effects on endotoxin-induced leukocyte adherence and plasma extravasation in postcapillary venules; Schmidt W et al.; BACKGROUND: C1-esterase inhibitor (C1-INH) has been shown to have beneficial effects in patients with sepsis . However, the microcirculatory effects of C1-INH during sepsis are unknown . This study investigated the influence of C1-INH on leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics in postcapillary venules of rat mesentery during endotoxemia . METHODS: Thirty-two anesthetized Wistar rats randomly received 1 of 4 treatments: pretreatment with infusion of C1-INH in a concentration of 7.5 U.kg-1 body weight (C1-INH-7.5 group, n = 8) or in a concentration of 15 U.kg-1 body weight (C1-INH-15 group, n = 8) followed by continuous infusion of Escherichia coli lipopolysaccharide (LPS) . The LPS group (n = 8) was pretreated with saline solution 30 minutes before LPS infusion . The control group (n = 8) received equivalent amounts of saline infusion . Leukocyte adherence, red blood cell velocity, and vessel diameters in postcapillary venules of rat mesentery were determined every 60 minutes during a period of 120 minutes using in vivo videomicroscopy . Vascular permeability was determined by measuring the extravasation of fluorescence-labeled albumin . Venular wall shear rate was calculated from mean red blood cell velocity and vessel diameter . RESULTS: LPS infusion induced a decrease in venular wall shear rate and an increase in leukocyte adherence and vascular permeability in postcapillary venules of rat mesentery . All microcirculatory disturbances were attenuated by pretreatment with C1-INH, showing no significant difference between the 2 concentrations . CONCLUSIONS: Pretreatment with C1-INH attenuates endotoxin-induced leukocyte adherence and macromolecular leakage in postcapillary venules of rat mesentery, indicating that complement inhibition might be a therapeutic tool in the treatment of sepsis. Biochim Biophys Acta, 1999 Feb 4, 1417(1), 37 - 50 A transfection compound series based on a versatile Tris linkage; Cameron FH et al.; The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants . The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group . The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties . We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents . A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity. Biochim Biophys Acta, 1999 Jan 27, 1410(1), 32 - 50 Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I; Osborne JP et al.; Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis . The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer . There are 20 conserved residues in subunit I and two in subunit II . Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E . coli . This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively . The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane . The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices . The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d . There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E . coli cytochrome bd-I numbering) . It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface . The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses . Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd. Nucleic Acids Res, 1999 Apr 1, 27(7), 1683 - 9 The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria; Tomita K et al.; It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNAAsn with the anticodon GUU . Nucleotide sequencing of starfish mt tRNAAsn revealed that the anticodon is GPsiU, U35 at the anticodon second position being modified to pseudouridine (Psi) . In contrast, mt tRNALys, corresponding to another lysine codon, AAG, has the anticodon CUU . mt tRNAs possessing anti-codons closely related to that of tRNAAsn, but responsible for decoding only two codons each-tRNAHis, tRNAAsp and tRNATyr-were found to possess unmodified U35 in all cases, suggesting the importance of Psi35 for decoding the three codons . Therefore, the decoding capabilities of two synthetic Escherichia coli tRNAAla variants with the anticodon GPsiU or GUU were examined using an E.coli in vitro translation system . Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was approximately 2-fold higher in the case of tRNAAlaGPsiU than tRNAAlaGUU . These findings imply that Psi35 of echinoderm mt tRNAAsn actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria. Nucleic Acids Res, 1999 Apr 1, 27(7), 1674 - 82 Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells; Wade-Martins R et al.; We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay . The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication . This element provides nuclear retention in cells expressing the EBV protein EBNA-1 . We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line . Episomal mitotic stability was quantitatively analysed using flow cytometry . The episomes were also tracked by time course photography of expanding colonies . A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth . Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days . Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies. EMBO J, 1999 Mar 15, 18(6), 1712 - 21 Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control; Lobner-Olesen A; A novel method was devised to measure the number of plasmids in individual Escherichia coli cells . With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured . Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes . When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased . At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected . This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome . However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome . At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome. EMBO J, 1999 Mar 15, 18(6), 1689 - 700 Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice; Harborth J et al.; NuMA is a nuclear matrix protein in interphase and relocates to the spindle poles in mitotis . Different NuMA constructs, in which either N- or C-terminal domains were deleted, and the full-length construct were expressed in Escherichia coli, and the NuMA polypeptides were purified to homogeneity and allowed to assemble in vitro . Electron microscopy showed that NuMA can build multiarm oligomers by interaction of the C-terminal globular domains . Each arm of the oligomer corresponds to a NuMA dimer . Oligomers with up to 10 or 12 arms have been observed for both full-length NuMA and for constructs that still contain the proximal part of the C-terminal tail domain . Other results from this laboratory have shown that transient overexpression of NuMA in HeLa cells induces a nuclear scaffold with a quasi-hexagonal organization that can fill the nuclei . Here we show that computer modelling of the three-dimensional packing of NuMA into such scaffolds can explain the different spacing of the hexagons seen when constructs with different coiled-coil lengths are used . Thus, the 12 arm oligomer, for which we have in vitro evidence, may be the structural unit from which the nuclear scaffold in transfected cells is built. EMBO J, 1999 Mar 15, 18(6), 1653 - 9 Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions; Brandi A et al.; The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA . We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer . The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable . A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium . The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein . Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance . The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA). EMBO J, 1999 Mar 15, 18(6), 1447 - 58 X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture; Raaijmakers H et al.; Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches . We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively . The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping . The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif . The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases . The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity . These include Asp40 and Asn62, which function as ligands for the bound calcium ions . A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations . A model for the complex with a four-way DNA junction is presented. EMBO J, 1999 Mar 15, 18(6), 1435 - 46 The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds; Hall DR et al.; The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum . We have determined the structure of ModE using multi-wavelength anomalous dispersion . Selenomethionyl and native ModE models are refined to 1 . 75 and 2.1 A, respectively and describe the architecture and structural detail of a complete transcriptional regulator . ModE is a homodimer and each subunit comprises N- and C-terminal domains . The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization . The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified . This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies . The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions . This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription . Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation. Biotechnol Appl Biochem, 1999 Apr, 29 ( Pt 2), 165 - 84 Mutational analysis of sickle haemoglobin (Hb) gelation; Li X et al.; The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures . We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced . The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients . Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells . The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization . These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate . This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants. Anal Biochem, 1999 Mar 15, 268(2), 343 - 53 Interactions of myristoylated alanine-rich C kinase substrate (MARCKS)-related protein with a novel solid-supported lipid membrane system (TRANSIL); Schmitz AA et al.; The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures . We have facilitated this analysis using a silica gel coated with a single phospholipid bilayer (TRANSIL) as the membranous phase . We demonstrate the validity of this method using MARCKS-related protein, a 20-kDa member of the MARCKS family (an acronym for myristoylated alanine-rich C kinase substrate) . The partition coefficients describing the association of unmyristoylated and myristoylated MAR