Biochemistry, 2000 Jun 27, 39(25), 7595 - 604 A novel delta(3),delta(2)-enoyl-CoA isomerase involved in the biosynthesis of the cyclohexanecarboxylic acid-derived moiety of the polyketide ansatrienin A; Patton SM et al.; The side chain of the antifungal polyketide ansatrienin A produced by Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC) derived moiety . This CHC in the coenzyme A activated form (CHC-CoA) is derived from shikimic acid via a pathway in which the penultimate step is the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA . We have purified a 28 kDa 2-cyclohexenylcarbonyl-CoA isomerase (ChcB) from S . collinus and cloned and sequenced the corresponding chcB gene . The predicted amino acid sequence of ChcB showed moderate sequence identity to members of the hydratase/isomerase superfamily of enzymes . The recombinant ChcB was overexpressed in Escherichia coli and purified to homogeneity using metal chelate chromatography . Kinetic analysis demonstrated that recombinant ChcB had wide substrate specificity and could catalyze a double bond isomerization using 2-cyclohexenylcarbonyl-CoA (K(m) 116 +/- 68 microM, k(cat)( )()3.7 +/- 1.0 min(-)(1)), trans-3-hexenyl-CoA (K(m) 39 +/- 10 microM, k(cat)( )()12.8 +/- 1 min(-)(1)), and vinylacetyl-CoA (K(m) 156 +/- 34 microM, k(cat)( )()29 +/- 3 min(-)(1)) as substrates . ChcB activity in cell extracts of S . collinus SP1, an insertionally disrupted chcB mutant, was shown to decrease by more than 99% (as compared to the wild-type strain) using all three of these substrates . The S . collinus SP1 strain, unlike the wild-type strain, could not produce omega-cyclohexyl fatty acids but was still able to grow efficiently on methyl oleate as a sole carbon source . These observations demonstrate that the S . collinus ChcB is required for catalyzing the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA during CHC-CoA biosynthesis but not for degradation of unsaturated fatty acids . The chcB gene does not appear to be associated with the ansatrienin biosynthetic gene cluster, which has previously been shown to contain at least one gene known to be essential for CHC-CoA biosynthesis . This finding represents a notable exception to the general rule regarding the clustering of polyketide biosynthetic pathway genes. Biochemistry, 2000 Jun 27, 39(25), 7546 - 51 13C and (15)N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant; Rishavy MA et al.; Heavy atom isotope effects at C-2, C-3, and the amino nitrogen of aspartate were determined for the reaction of porcine heart cytosolic aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase . The effects of deuteration at C-2 of aspartate and of D(2)O on the observed heavy atom isotope effects were determined . The multiple isotope effects support the contribution of C(alpha)-H cleavage, ketimine hydrolysis, and oxaloacetate dissociation to the rate limitation with the wild-type enzyme . The existence of a quinonoid intermediate could not be determined due to the kinetic complexity of the enzyme . For the tyrosine-225 to phenylalanine mutant, we are able to conclude that ketimine hydrolysis is the major rate-determining step. Biochemistry, 2000 Jun 27, 39(25), 7501 - 7 Inhibition of p-hydroxyphenylpyruvate dioxygenase by the diketonitrile of isoxaflutole: a case of half-site reactivity; Garcia I et al.; p-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from p-hydroxyphenylpyruvate and molecular oxygen . In plants, this enzyme is the molecular target of new families of very active bleaching herbicides . In the study presented here, we report for the first time on the purification to homogeneity of a plant enzyme, as obtained from recombinant Escherichia coli cells expressing a cDNA encoding carrot HPPD . The purified enzyme allowed us to carry out a detailed characterization of the inhibitory properties of a diketonitile (DKN), the active inhibitor formed from the benzoylisoxazole herbicide isoxaflutole . Inhibition kinetic analyses confirmed that DKN exerts a slow and tight-binding inhibition of HPPD, competitive with respect to the p-hydroxyphenylpyruvate substrate . The stoichiometry of DKN binding to HPPD determined by kinetic analyses or by direct binding of {(14)C}DKN revealed a half-site reactivity of DKN. Biochemistry, 2000 Jun 27, 39(25), 7492 - 500 Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase; Contestabile R et al.; Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme . In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine . To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65 . The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group . The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined . The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed . However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate . The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine . These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure. Biochemistry, 2000 Jun 27, 39(25), 7414 - 9 Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue; Graversen JH et al.; Kringle domains are found in a number of proteins where they govern protein-protein interactions . These interactions are often sensitive to lysine and lysine analogues, and the kringle-lysine interaction has been used as a model system for investigating kringle-protein interactions . In this study, we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry . We find that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin . Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin . We also find that Asp 57 and Asp 55 of plasminogen kringle 4, which both were found to interact with the low molecular weight ligand with an almost identical geometry in the crystal of the complex, are not of equal functional importance in t-AMCHA binding . Mutating Asp 57 to an Asn totally eliminates binding, whereas the Asp 55 to Asn, like the Arg 71 to Gln mutation, was found only to decrease affinity. Biochemistry, 2000 Jun 27, 39(25), 7406 - 13 Conformation of the isolated cepsilon3 domain of IgE and its complex with the high-affinity receptor, FcepsilonRI; Henry AJ et al.; Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcepsilonRI, on the surface of mast cells and basophils . Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (Cepsilon3) in binding to FcepsilonRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cepsilon3 and Cepsilon4 domains . We have expressed the isolated Cepsilon3 in Escherichia coli, measured its affinity for FcepsilonRI, and examined its conformation alone and in the complex with FcepsilonRI . Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer . The kinetics of binding to an immobilized fragment of the FcepsilonRI alpha-chain, measured by surface plasmon resonance, yields an affinity constant K(a) = 5 x 10(6) M(-)(1), as compared with 4 x 10(9) M(-)(1) for IgE . The circular dichroism spectrum and measurements of fluorescence as a function of the concentration of a denaturant do not reveal any recognizable secondary structure or hydrophobic core . On binding to the FcepsilonRI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cepsilon3 is unchanged in the complex . Thus the isolated Cepsilon3 domain is sufficient for binding to FcepsilonRI, but with lower affinity than IgE . This may be due to the loss of its native immunoglobulin domain structure or to the requirement for two Cepsilon3 domains to constitute the complete binding site for FcepsilonRI or to a combination of these factors. Biochemistry, 2000 Jun 27, 39(25), 7380 - 7 The tissue factor region that interacts with substrates factor IX and Factor X; Kirchhofer D et al.; The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF) . TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain . We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation . Soluble TF (sTF) mutants were expressed in E . coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes . The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface . According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204 . Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction . Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding . The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa . Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis. Biochemistry, 2000 Jun 27, 39(25), 7331 - 6 Structural similarities between MutT and the C-terminal domain of MutY; Volk DE et al.; One of the functions of MutY from Escherchia coli is removal of adenine mispaired with 7,8-dihydro-8-oxoguanine (8-oxoG), a common lesion in oxidatively damaged DNA . MutY is composed of two domains: the larger N-terminal domain (p26) contains the catalytic properties of the enzyme while the C-terminal domain (p13) affects substrate recognition and enzyme turnover . On the basis of sequence analyses, it has been recently suggested that the C-terminal domain is distantly related to MutT, a dNTPase which hydrolyzes 8-oxo-dGTP {Noll et al . (1999) Biochemistry 38, 6374-6379} . We have studied the solution structure of the C-terminal domain of MutY by NMR and find striking similarity with the reported solution structure of MutT . Despite low sequence identity between the two proteins, they have similar secondary structure and topology . The C-terminal domain of MutY is composed of two alpha-helices and five beta-strands . The NOESY data indicate that the protein has two beta-sheets . MutT is also a mixed alpha/beta protein with two helices and two beta-sheets composed of five strands . The secondary structure elements are similarly arranged in the two proteins. FEBS Lett, 2000 Jun 16, 475(2), 135 - 8 1H, 15N and (13)C assignments and secondary structure of the EGF-like module pair 3-4 from vitamin K-dependent protein S; Muranyi A et al.; Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity . The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property . Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4 . In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained . The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation . Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet. Biochemistry, 2000 Jun 13, 39(23), 6864 - 73 Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k); Berggard T et al.; Calbindin D(28k) is a member of a large family of intracellular Ca(2+) binding proteins characterized by EF-hand structural motifs . Some of these proteins are classified as Ca(2+)-sensor proteins, since they are involved in transducing intracellular Ca(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca(2+) binding . The hydrophobic patch serves as an interaction site for target enzymes . Other members of this group are classified as Ca(2+)-buffering proteins, because they remain closed after Ca(2+) binding and participate in Ca(2+) buffering and transport functions . ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca(2+)-free and Ca(2+)-loaded form of calbindin D(28k) have exposed hydrophobic surfaces . Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D(28k) most likely interacts with other cellular components in vivo . A Ca(2+)-induced conformational change was readily detected by several optical spectroscopic methods . Thus, calbindin D(28k) shares some of the properties of Ca(2+)-sensor proteins . However, the Ca(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin . The data also shows that calbindin D(28k) undergoes a rapid and reversible conformational change in response to a H(+) concentration increase within the physiological pH range . The pH-dependent conformational change was shown to reside mainly in EF-hands 1-3 . Urea-induced unfolding of the protein at pH 6, 7, and 8 showed that the stability of calbindin D(28k) was increased in response to H(+) in the range examined . The results suggest that calbindin D(28k) may interact with targets in a Ca(2+)- and H(+)-dependent manner. Infect Immun, 2000 Jul, 68(7), 4363 - 7 Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages; Lai XH et al.; Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype . We show here that highly purified ClyA and ClyA-expressing E . coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages. Infect Immun, 2000 Jul, 68(7), 4344 - 8 Mechanical fractionation reveals structural requirements for enteropathogenic Escherichia coli Tir insertion into host membranes; Gauthier A et al.; Enteropathogenic Escherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system . Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells . In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins . We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation . This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin. Infect Immun, 2000 Jul, 68(7), 4064 - 74 Full capacity of recombinant Escherichia coli heat-stable enterotoxin fusion proteins for extracellular secretion, antigenicity, disulfide bond formation, and activity; Batisson I et al.; We have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length . However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported . To address this possibility, we genetically fused peptides containing all or part of the E . coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG . Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E . coli cells in a heat-stable active oxidized (disulfide-bonded) form . Indeed, in contrast to many earlier studies, blocking the natural NH(2) or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity . Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner . These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition) . Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E . coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis . None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E . coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins . In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway . In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E . coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin. Infect Immun, 2000 Jul, 68(7), 3956 - 64 Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein; Boguslavsky S et al.; A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum . The pvpA gene was cloned, expressed in Escherichia coli, and sequenced . It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively . In addition, 50% homology was found with the MGC2 cytadhesin of M . gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M . pneumoniae . The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times . Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation . The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon . Size variation of the PvpA protein was observed among M . gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences . By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure . Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M . gallisepticum. Infect Immun, 2000 Jul, 68(7), 3941 - 8 Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica; Kim TS et al.; The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized . The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa . An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD) . The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5 . This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis . The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms . An F . hepatica cDNA library was screened with the SOD gene fragment as a probe . As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined . The gene had an open reading frame of 438 bp and 146 deduced amino acids . Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies . The coding region of the F . hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli . Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide . This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD . The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects. Inflamm Res, 2000 Apr, 49(4), 170 - 6 Participation of endogenous endothelins in delayed eosinophil and neutrophil recruitment in mouse pleurisy; Sampaio AL et al.; OBJECTIVE: Investigate the role of endothelins in leukocyte recruitment in allergic and non allergic inflammation . METHODS: Pleurisy was induced in mice by intrathoracic injection of ovalbumin (OVA; in sensitized animals), E . coli LPS, carrageenan, Mycobacterium bovis (BCG) or zymosan . Animals were treated with BQ-123 or BQ-788 (1.5-150 pmol/cavity), or intravenously with bosentan (30 mg/kg) . RESULTS: None of the ET receptor antagonists modified early neutrophil recruitment (at 4 h) induced by OVA, LPS, carrageenan, BCG or zymosan or plasma leakage caused by carrageenan or zymosan . Mononuclear and eosinophil accumulation triggered by OVA were reduced by BQ-123 (150 pmol/cavity) or bosentan (68 and 43% inhibition of eosinophilia), but unaffected by BQ-788 . BQ-123 and bosentan also inhibited LPS increases in neutrophil (by 67 and 40%) and eosinophil (by 63 and 74%) at 24 h . CONCLUSIONS: Endothelins, acting via ETA receptors, play a role in late eosinophil and neutrophil accumulation (24 h), but not in the acute (4 h) neutrophilic response. MMWR Morb Mortal Wkly Rep, 2000 Apr 21, 49(15), 321 - 4 Escherichia coli O111:H8 outbreak among teenage campers--Texas, 1999; Copper ions mediate the lethality induced by hydrogen peroxide in low iron conditions in Escherichia coli; Comissao Nacional de Energia Nuclear, Diretoria de Radioprotecao e Seguranca, Superintendencia de Licenciamento e Controle, rua General Severiano, 90, Botafogo, CEP 22294-900, RJ, Rio de Janeiro, BrazilIron ions mediate the formation of lethal DNA damage by hydrogen peroxide . However, when cells are depleted of iron ions by the treatment with iron chelators, DNA damage can still be detected . Here we show that the formation of such damage in low iron conditions is due to the participation of copper ions . Copper chelators can inhibit cell inactivation, DNA strand breakage and mutagenesis induced by hydrogen peroxide in cells pre-treated with iron chelators . The Fpg and UvrA proteins play an important role in the repair of DNA lesions formed in these conditions, as suggested by the great sensitivity of the uvrA and fpg mutant strains to the treatment when compared to the wild type strain. J Virol Methods, 2000 Jun, 87(1-2), 53 - 62 Characterization of a strain-specific monoclonal antibody to hepatitis delta virus antigen; Hsu SC et al.; Sequences of the hepatitis delta virus (HDV) vary to different degrees among isolates . A monoclonal antibody, designated as HP6A1, against the antigen of HDV (HDAg) has been characterized for its specificity . HP6A1 bound to HDAg of isolate 25 (genotype I) that was used for immunization, but not to others of both genotypes I and II . The epitope recognized by HP6A1 was then determined by a phage library displaying various heptapeptides . A consensus peptide deduced has the best match with that of residues 4-10 of HDAg (isolate 25) . To confirm the phage mapping result, Escherichia coli recombinant proteins containing different lengths and various segments of HDAg (isolate 25) were constructed . The shortest HDAg segment contained in the fusion protein that reacted with HP6A1 was residues 1-10 . When this peptide was added to the N-terminus of a heterologous protein engineered for eucaryotic expression, the fusion protein was detected by HP6A1 . It is concluded that HP6A1 recognizes an epitope located at the N-terminus of HDAg (isolate 25) . Since viruses of quasi-species exist in natural infections, a question of how different viral strains interact in vivo remains to be explored . The highly specific MAb opens a possibility to examine the fate of one strain in the presence of other related species in a cell transfection system. FEMS Microbiol Lett, 2000 Jun 15, 187(2), 133 - 8 Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility; Pembroke JT et al.; The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements . Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA . The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently . Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA . When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb . When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated . Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship . These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied . This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation. FEMS Microbiol Lett, 2000 Jun 15, 187(2), 127 - 32 The FAPY-DNA glycosylase (Fpg) is required for survival of the cyanobacterium Synechococcus elongatus under high light irradiance; Muhlenhoff U; The gene for the DNA repair enzyme Fpg from Synechococcus elongatus was detected immediately downstream of the photosystem I gene psaE . fpg is likely expressed together with psaE by transcriptional readover while psaE is mostly expressed independently . Segregated psaE and fpg deletion strains were obtained upon insertional inactivation of both genes in S . elongatus . These mutants are viable under photoautotrophic conditions, but fail to grow under high light regimes that likely cause oxidative stress . These high light sensitive phenotypes suggest that the Fpg protein, which has been shown to repair DNA lesions caused by reactive oxygen species in Escherichia coli, may be involved in the photoprotection of cyanobacteria against oxidative damage caused under high irradiance. FEMS Microbiol Lett, 2000 Jun 15, 187(2), 115 - 22 Regulation of the divergent guaBA and xseA promoters of Escherichia coli by the cyclic AMP receptor protein; Hutchings MI et al.; The gua promoter (guaP) of Escherichia coli resembles those for ribosomal RNA (rrn) operons and lies in a close back-to-back arrangement with the promoter for xseA (xseP) . Transcription from guaP is subject to stringent control and growth-rate-dependent regulation, and to repression by DnaA and PurR . In addition, transcription from guaP is regulated by the cyclic AMP receptor protein (CRP) . Plasmid-borne promoter fusions to the receptor gene for chloramphenicol acetyl transferase were used to assess the role of CRP in controlling transcription from guaP and xseP following a downshift of cultures from rich into minimal medium . CRP is required to activate guaBA transcription and repress xseA transcription following downshift . Bandshift assays with a DNA fragment carrying the divergent promoters revealed specific binding of CRP . We propose that CRP, binding to a near-consensus site centred at -117.5, activates transcription from guaP and obstructs transcription from the xseA promoter. J Biol Chem, 2000 Aug 18, 275(33), 25445 - 50 Structure of GATE-16, membrane transport modulator and mammalian ortholog of autophagocytosis factor Aut7p; Paz Y et al.; The GATE-16 protein participates in intra-Golgi transport and can associate with the N-ethylmaleimide-sensitive fusion protein and with Golgi SNAREs . The yeast ortholog of GATE-16 is the autophagocytosis factor Aut7p . GATE-16 is also closely related to the GABA receptor-associated protein (GABARAP), which has been proposed to cluster neurotransmitter receptors by mediating interaction with the cytoskeleton, and to the light chain-3 subunit of the neuronal microtubule-associated protein complex . Here, we present the crystal structure of GATE-16 refined to 1.8 A resolution . GATE-16 contains a ubiquitin fold decorated by two additional N-terminal helices . Proteins with strong structural similarity but no detectable sequence homology to GATE-16 include Ras effectors that mediate diverse downstream functions, but each interacts with Ras by forming pseudo-continuous beta-sheets . The GATE-16 surface suggests that it binds its targets in a similar manner . Moreover, a second potential protein-protein interaction site on GATE-16 may explain the adapter activity observed for members of the GATE-16 family. EMBO J, 2000 Jun 15, 19(12), 3038 - 48 Conservation of sigma-core RNA polymerase proximity relationships between the enhancer-independent and enhancer-dependent sigma classes; Wigneshweraraj SR et al.; Two distinct classes of RNA polymerase sigma factors (sigma) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation . Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent sigma(54), we mapped several sites of proximity to the beta and beta' subunits of the core RNA polymerase . Remarkably, most sites localized to those previously identified as close to the enhancer-independent sigma(70) and sigma(38) . This indicates a common use of sets of sequences in core for interacting with the two sigma classes . Some sites chosen in sigma(54) for modification with Fe-BABE were positions, which when mutated, deregulate the sigma(54)-holoenzyme and allow activator-independent initiation and holoenzyme isomerization . We infer that these sites in sigma(54) may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex . One site of sigma(54) proximity to the core is apparently not evident with sigma(70), and may represent a specialized interaction. EMBO J, 2000 Jun 15, 19(12), 3028 - 37 sigma factor selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and lrp transcription factors; Colland F et al.; osmY is a stationary phase-induced and osmotically regulated gene in Escherichia coli that requires the stationary phase RNA polymerase (Esigma(S)) for in vivo expression . We show here that the major RNA polymerase, Esigma(70), also transcribes osmY in vitro and, depending on genetic background, even in vivo . The cAMP receptor protein (CRP) bound to cAMP, the leucine-responsive regulatory protein (Lrp) and the integration host factor (IHF) inhibit transcription initiation at the osmY promoter . The binding site for CRP is centred at -12.5 from the transcription start site, whereas Lrp covers the whole promoter region . The site for IHF maps in the -90 region . By mobility shift assay, permanganate reactivity and in vitro transcription experiments, we show that repression is much stronger with Esigma(70) than with Esigma(S) holoenzyme . We conclude that CRP, Lrp and IHF inhibit open complex formation more efficiently with Esigma(70) than with Esigma(S) . This different ability of the two holoenzymes to interact productively with promoters once assembled in complex nucleoprotein structures may be a crucial factor in generating sigma(S) selectivity in vivo. Fertil Steril, 2000 Jun, 73(6), 1250 - 2 Vertebral osteomyelitis: a rare complication of transvaginal ultrasound-guided oocyte retrieval; Almog B et al.; OBJECTIVE: To present a case of vertebral osteomyelitis as a complication of transvaginal oocyte retrieval . DESIGN: Case report . SETTING: The IVF unit of a university-affiliated hospital . PATIENT(s): A 41-year-old woman who underwent IVF-ET treatment . INTERVENTION(s): Standard IVF-ET treatment cycles with the use of transvaginal ultrasound for oocyte retrieval and computed tomography-guided needle aspiration . MAIN OUTCOME MEASURE(s): Recovery of the patient, sequelae, and recurrence . RESULT(s): Vertebral osteomyelitis was diagnosed and treated with antibiotics . CONCLUSION(s): When severe low back pain occurs after ovum retrieval, vertebral osteomyelitis should be considered . Early diagnosis requires a high index of suspicion. Appl Microbiol Biotechnol, 2000 May, 53(5), 536 - 41 On-line monitoring of growth of Escherichia coli in batch cultures by bioluminescence; Marincs F; Bioluminescence was used to monitor growth of Escherichia coli in batch cultures on-line . Light emission of a strain engineered for constitutive bioluminescence was monitored with a simple set-up consisting of a photodiode, a photodetector amplifier and a recorder . Bioluminescence and colony forming units (CFU) of the cultures increased and decreased proportionally and were correlated during every growth phase at temperatures between 28 degrees C and 40 degrees C . Up to the late log (deceleration) phase, both light emission and CFU increased rapidly . Beyond the stationary phase these characteristics decreased very slowly at lower temperatures, while at higher ones they declined more rapidly . Towards the end of the cultivation, light emission of the cultures dropped to undetectable levels, even though CFU were recovered . This was particularly marked at lower temperatures where non-luminescent cultures retained very high CFU . This indicates that the actual metabolism of cells in a culture can be at a very low level or completely shut down, yet cells retain their capability to be culturable . The on-line technology described here has a number of potential uses in the laboratory and industry. J Immunol Methods, 2000 Jun 23, 240(1-2), 165 - 83 A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody; Boldicke T et al.; A monoclonal anti-peptide antibody (2E11) was raised against the synthetic peptide 38 (C-L-D-K-S-G-L-P-S-D-R-F-F-A) representing a part of the variable region of the Vbeta 6.2 T-cell receptor . This mAb (IgG(1), kappa light chain) bound very specifically to peptide 38 as shown by ELISA but did not recognize the corresponding native Vbeta 6.2 T-cell receptor on T-cells . For epitope analysis, overlapping peptides of 4-10 amino acids in length corresponding to the sequence of peptide 38 were synthesized and assayed by SPOT synthesis on cellulose sheets . The shortest peptide recognized was L-P-S-D-R . The specificity of mAb 2E11 was examined with 100 different peptides comprising other parts of the different variable Vbeta domains of the human T-cell receptor that do not include the epitope region L-P-S-D-R . None of these peptides were recognized . The chemical synthesis of a peptide with the sequence L-P-S-D-R on Sepharose beads allowed to efficiently purify the mAb 2E11 in a single step by affinity chromatography . An equilibrium binding constant of 4.9x10(6) l/mol was determined for mAb 2E11 by using rhodamine-green-labelled peptide 38 in fluorescence correlation spectroscopy . In order to demonstrate that peptide 38 can be used as an affinity-tag, it was fused to the carboxyl-terminus of interferon regulatory factor-1 (IRF-1) . It could be shown that in vitro translated peptide 38 tagged IRF-1 was immunoprecipitated by the mAb 2E11 and that the fusion protein could be purified by immunoaffinity chromatography . Additionally peptide 38 was fused to the amino-terminus of the Taq polymerase . This recombinant protein was expressed in E . coli and specifically detected in a Dot blot and Western blot using mAb 2E11. J Biol Chem, 2000 Aug 25, 275(34), 26607 - 14 Identification of a glutamic acid and an aspartic acid residue essential for catalytic activity of aspergillopepsin II, a non-pepsin type acid proteinase; Huang XP et al.; Aspergillopepsin II from Aspergillus niger var . macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase . To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis . The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro . The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions . Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants . The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions . The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded . In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions . Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II. J Biol Chem, 2000 Aug 25, 275(34), 26556 - 65 Acute cadmium exposure inactivates thioltransferase (Glutaredoxin), inhibits intracellular reduction of protein-glutathionyl-mixed disulfides, and initiates apoptosis; Chrestensen CA et al.; Oxidative stress broadly impacts cells, initiating regulatory pathways as well as apoptosis and necrosis . A key molecular event is protein S-glutathionylation, and thioltransferase (glutaredoxin) is a specific and efficient catalyst of protein-SSG reduction . In this study 30-min exposure of H9 and Jurkat cells to cadmium inhibited intracellular protein-SSG reduction, and this correlated with inhibition of the thioltransferase system, consistent with thioltransferase being the primary intracellular catalyst of deglutathionylation . The thioredoxin system contributed very little to total deglutathionylase activity . Thioltransferase and GSSG reductase in situ displayed similar dose-response curves (50% inhibition near 10 micrometer cadmium in extracellular buffer) . Acute cadmium exposure also initiated apoptosis, with H9 cells being more sensitive than Jurkat . Moreover, transfection with antisense thioltransferase cDNA was incompatible with cell survival . Collectively, these data suggest that thioltransferase has a vital role in sulfhydryl homeostasis and cell survival . In separate experiments, cadmium inhibited the isolated component enzymes of the thioltransferase and thioredoxin systems, consistent with the vicinal dithiol nature of their active sites: thioltransferase (IC(50) approximately 1 micrometer), GSSG reductase (IC(50) approximately 1 micrometer), thioredoxin (IC(50) approximately 8 micrometer), thioredoxin reductase (IC(50) approximately 0.2 micrometer) . Disruption of the vicinal dithiol on thioltransferase (via oxidation to C22-SS-C25; or C25S mutation) protected against cadmium, consistent with a dithiol chelation mechanism of inactivation. FEBS Lett, 2000 Jun 9, 475(1), 35 - 8 Catalase activity of oxygenase domain of rat neuronal nitric oxide synthase . Evidence for product formation from L-arginine; Adhikari S et al.; Nitric oxide synthases (NOSs) catalyze the formation of nitric oxide from L-arginine . We purified the heme containing, tetrahydrobiopterin-free, oxygenase domain of rat neuronal nitric oxide synthase (nNOSox) overexpressed in Escherichia coli . We found catalase activity in nNOSox . This is significant because H(2)O(2) may also be a product of nitric oxide synthases . We found H(2)O(2) assisted product formation from N-hydroxy-L-arginine and even from L-arginine both in the presence and in absence of tetrahydrobiopterin . We propose how heme moiety of the oxygenase domain alone is sufficient to carry out both steps of the NOS catalysis. Gene, 2000 May 30, 250(1-2), 201 - 8 Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast; Annab LA et al.; The BRCA1 gene, mutations of which contribute significantly to hereditary breast cancer, was not identified in the existing YAC and BAC libraries . The gene is now available only as a set of overlapping fragments that form a contig . In this work we describe direct isolation of a genomic copy of BRCA1 from human DNA by transformation-associated recombination (TAR) cloning . Despite the presence of multiple repeats, most of the primary BRCA1 YAC isolates did not contain detectable deletions and could be stably propagated in a host strain with conditional RAD52 . Similar to other circular YACs, approximately 90kb BRCA1 YACs were efficiently and accurately retrofitted into bacterial artificial chromosomes (BACs) with the Neo(R) mammalian selectable marker and transferred as circular BAC/YACs in E . coli cells . The BRCA1 BAC/YAC DNAs were isolated from bacterial cells and were used to transfect mouse cells using the Neo(R) gene as selectable marker . Western blot analysis of transfectants showed that BRCA1 YACs isolated by a TAR cloning contained a functional gene . The advantage of this expression vector is that the expression of BRCA1 is generated from its own regulatory elements and does not require additional promoter elements that may result in overexpression of the protein . In contrast to the results with cDNA expression vectors, the level of BRCA1 expression from this TAR vector is stable, does not induce cell death, maintains serum regulation, and approximates the level of endogenously expressed BRCA1 in human cells . The entire isolation procedure of BRCA1 described in this paper can be accomplished in approximately 10 days and can be applied to isolation of gene from clinical material . We propose that the opportunity to directly isolate normal and mutant forms of BRCA1 will greatly facilitate analysis of the gene and its contribution to breast cancer. Eur J Hum Genet, 2000 May, 8(5), 331 - 8 Importance of searching for associated mitochondrial DNA alterations in patients with multiple deletions; Paul R et al.; Multiple mitochondrial DNA (mtDNA) deletions have been reported in patients with autosomal dominant and recessive disorders . We studied several affected and one non-affected individuals belonging to a pedigree in which the inheritance of the pathological trait was compatible with an autosomique dominant transmission . Affected members had late-onset multisystem disorders with multiple mtDNA deletions in skeletal muscle . But this family presented a striking difference from previously described cases, because none of the patients had progressive external ophthalmoplegia (PEO) . We also studied one young boy with a no contributary family history . He had a cerebellar ataxia with PEO and multiple mtDNA deletions in muscle . Molecular analysis revealed that in the first family, repeated sequences were present at the breakpoint junctions, whereas such motifs were not found in the young patient's case . In the first family, we evidenced mtDNA point mutations in clones containing breakpoint junctions and a 9-bp motif triplication in the intergenic COII/tRNA(Lys) region, whereas this sequence is repeated twice in the wild type mtDNA . Our results suggest that multiple deletions observed in the two pedigrees result from different molecular mechanisms and point out the role of repeated sequences in the first pedigree . No mtDNA repair system has been described in mammals so far, but the molecular abnormalities found in the first family suggest that a defect in an mtDNA repair system, homologous to the E . coli MutHLS pathway, could be responsible for such a phenotype. Exp Cell Res, 2000 May 25, 257(1), 206 - 12 Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase; Escalas N et al.; HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition . We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase . Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint . We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity . This effect can be counteracted by coexpression of the WEE1 kinase . In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis . The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed. Graefes Arch Clin Exp Ophthalmol, 2000 Apr, 238(4), 359 - 65 Involvement of superoxide generated by polymorphonuclear leukocytes in endotoxin-induced uveitis; Hashida M et al.; BACKGROUND: Although superoxide is thought to be involved in the development of endotoxin-induced uveitis (EIU), the role of superoxide generation by polymorphonuclear leukocytes (PMNs) has not been fully elucidated . The purpose of this study was to investigate the role of peripheral blood PMNs in the development of EIU . METHODS: EIU was induced in Lewis rats by injection of lipopolysaccharide (LPS) in one hind footpad . Superoxide generation was assayed by measuring the reduction of ferricytochrome c (cyt c) . EIU severity was assessed by histological examination, and the relationship between the injected dose of LPS in vivo and the intensity of superoxide generation by peripheral PMNs or intraocular PMNs was studied . Twenty-four hours after the injection of LPS (2, 20, or 200 microg/rat), peripheral blood PMNs were collected and stimulated with phorbol 12-myristate 13-acetate (PMA) . The time course of superoxide generation by PMNs after LPS injection (3, 6, 12, 24, 48, and 72 h) was also investigated . To test the possible inhibition of superoxide generation by protein kinase C (PKC) inhibitors, H-7 and staurosporine were added for the incubation . In addition to the measurement of cyt c reduction, western blotting was used to detect PKC activity . The direct effect of LPS on PMNs was tested by priming naive PMNs with LPS in vitro . RESULTS: The intensity of superoxide generation by PMNs and the severity of EIU were dependent on the dose of injected LPS . No apparent superoxide generation was detected from intraocular PMNs . The time course of superoxide generation was similar to that of EIU severity . H-7 or staurosporine inhibited superoxide generation dose dependently and suppressed phosphorylation of PKC . Priming with LPS in vitro prompted minimal superoxide generation by naive PMNs . CONCLUSION: Superoxide generation by peripheral blood PMNs but not by intraocular PMNs from rats with EIU was demonstrated, and it is suggested that superoxide generation by PKC cascade might be involved in the pathogenesis of EIU. Bioorg Med Chem Lett, 2000 May 1, 10(9), 907 - 10 Incorporation of 4-thiothymidine into DNA by the Klenow fragment and HIV-1 reverse transcriptase; Rao TV et al.; The 5'-triphosphate of 4-thiothymidine (4S-TTP) is an excellent substrate for the Klenow fragment of Escherichia coli DNA polymerase 1 and HIV-1 reverse transcriptase with values of k(cat)/Km within a factor of approximately 3 of those for TTP . A large UV change (deltaepsilon= -9770 M(-1)cm(-1) at 340 nm) associated with incorporation of 4S-TMP into nucleic acid duplexes makes possible a rapid, continuous spectrophotometric assay of the reaction progress. Rheumatology (Oxford), 2000 May, 39(5), 537 - 41 Geographic clustering of an outer surface protein A mutant of Borrelia burgdorferi . Possible implications of multiple variants for Lyme disease persistence; Malawista SE et al.; DNA sequences encoding full-length outer surface protein (Osp) A were amplified from four joint fluid samples over 4.5 months from a patient with chronic Lyme arthritis, with a variant from wild type only found in sample 3 . Rather than a mutation in vivo, these findings suggested a mixed infection in which BORRELIA: containing the wild-type and mutant ospA were waxing and waning in the patient's joint . If so, we reasoned that the mutant should be present in the community . We therefore took the novel epitope resulting from the mutation, expressed as a fusion protein in Escherichia coli, and performed Western blots on 80 high-titred stored sera; however, all except that of our index patient were negative . We then collected 36 stored sera from patients with Lyme disease residing within 10 miles of where the index patient had lived . An additional two sera from this circumscribed area were positive (P = 0.038) . These findings show that results from single samples can be misleading, and suggest that the OspAs expressed in force late in Lyme arthritis are the same ones introduced initially into the host . Moreover, they allow a speculative mechanism for disease persistence not previously considered, in which antigenically distinct B . burgdorferi variant proteins present themselves serially to the immune system. Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7190 - 5 Adenosylcobalamin inhibits ribosome binding to btuB RNA; Nou X et al.; Expression of the btuB gene encoding the outer membrane cobalamin transporter in Escherichia coli is strongly reduced on growth with cobalamins . Previous studies have shown that this regulation occurs in response to adenosylcobalamin (Ado-Cbl) and operates primarily at the translational level . Changes in the level and stability of btuB RNA are consequences of the modulated translation initiation . To examine how Ado-Cbl affects translation, the binding of E . coli 30S ribosomal subunits to btuB RNA was investigated by using a primer extension inhibition assay . Ribosome binding to btuB RNA was much less efficient than to other RNAs and was preferentially lost when the ribosomes were subjected to a high-salt wash . Ribosome binding to btuB RNA was inhibited by Ado-Cbl but not by cyanocobalamin, with half-maximal inhibition around 0.3 microM Ado-Cbl . Ribosome-binding activity was increased or decreased by mutations in the btuB leader region, which affected two predicted RNA hairpins and altered expression of btuB-lacZ reporters . Finally, the presence of Ado-Cbl elicited formation of a single primer extension-inhibition product with the same specificity and Cbl-concentration dependence as the inhibition of ribosome binding . These results indicate that btuB expression is controlled by the specific binding of Ado-Cbl to btuB RNA, which then affects access to its ribosome-binding sequence. Plant Cell, 2000 Jun, 12(6), 991 - 1002 Arabidopsis MutS homologs-AtMSH2, AtMSH3, AtMSH6, and a novel AtMSH7-form three distinct protein heterodimers with different specificities for mismatched DNA; Culligan KM et al.; Arabidopsis mismatch repair genes predict MutS-like proteins remarkably similar to eukaryotic MutS homologs-MSH2, MSH3, and MSH6 . A novel feature in Arabidopsis is the presence of two MSH6-like proteins, designated AtMSH6 and AtMSH7 . Combinations of Arabidopsis AtMSH2 with AtMSH3, AtMSH6, or AtMSH7 proteins-products of in vitro transcription and translation-were analyzed for interactions by analytical gel filtration chromatography . The AtMSH2 protein formed heterodimers with AtMSH3, AtMSH6, and AtMSH7, but no single proteins formed homodimers . The abilities of the various heterodimers to bind to mismatched 51-mer duplexes were measured by electrophoretic mobility-shift assays . Similar to the behavior of the corresponding human proteins, AtMSH2*AtMSH3 heterodimers bound "insertion-deletion" DNA with three nucleotides (+AAG) or one nucleotide (+T) looped out much better than they bound DNA with a base/base mispair (T/G), whereas AtMSH2*AtMSH6 bound the (+T) substrate strongly, (T/G) well, and (+AAG) no better than it did a (T/A) homoduplex . However, AtMSH2*AtMSH7 showed a different specificity: moderate affinity for a (T/G) substrate and weak binding of (+T) . Thus, AtMSH2*AtMSH7 may be specialized for lesions/base mispairs not tested or for (T/G) mispairs in special contexts. Plant Cell, 2000 Jun, 12(6), 949 - 61 Developmental regulation of methyl benzoate biosynthesis and emission in snapdragon flowers; Dudareva N et al.; In snapdragon flowers, the volatile ester methyl benzoate is the most abundant scent compound . It is synthesized by and emitted from only the upper and lower lobes of petals, where pollinators (bumblebees) come in contact with the flower . Emission of methyl benzoate occurs in a rhythmic manner, with maximum emission during the day, which correlates with pollinator activity . A novel S-adenosyl-l-methionine:benzoic acid carboxyl methyl transferase (BAMT), the final enzyme in the biosynthesis of methyl benzoate, and its corresponding cDNA have been isolated and characterized . The complete amino acid sequence of the BAMT protein has only low levels of sequence similarity to other previously characterized proteins, including plant O-methyl transferases . During the life span of the flower, the levels of methyl benzoate emission, BAMT activity, BAMT gene expression, and the amounts of BAMT protein and benzoic acid are developmentally and differentially regulated . Linear regression analysis revealed that production of methyl benzoate is regulated by the amount of benzoic acid and the amount of BAMT protein, which in turn is regulated at the transcriptional level. Plant Cell, 2000 Jun, 12(6), 853 - 70 Sterol methyltransferase 1 controls the level of cholesterol in plants; Diener AC et al.; The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol . The ethyl addition is the product of two sequential methyl additions . Arabidopsis contains three genes-sterol methyltransferase 1 (SMT1), SMT2, and SMT3-homologous to yeast ERG6, which is known to encode an S-adenosylmethionine-dependent C-24 SMT that catalyzes a single methyl addition . The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p . Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant . The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis . smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content . Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics . The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis . The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition. J Biol Chem, 2000 Aug 25, 275(34), 25879 - 82 Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog; Lukyanov KA et al.; It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group . The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification . Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata . Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm) . The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light . The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein . Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm . They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins. J Bacteriol, 2000 Jun, 182(12), 3590 - 2 In vivo splicing and functional characterization of Mycobacterium leprae RecA; Frischkorn K et al.; The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins . In contrast to the M . tuberculosis RecA, the M . leprae RecA is not spliced in Escherichia coli . We demonstrate here that M . leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination. J Bacteriol, 2000 Jun, 182(12), 3544 - 52 Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli; Boesch KC et al.; The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism . Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization . To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants . Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene . Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer . Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically . All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ . There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate . In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein. J Bacteriol, 2000 Jun, 182(12), 3529 - 35 Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon; Holcroft CC et al.; The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose . Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the -35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at -92.5) . We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation . Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions . We also expressed a derivative of the alpha subunit of RNA polymerase deleted for the entire C-terminal domain (alpha-Delta235) and assayed expression from rhaBAD promoter fusions . The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold) . Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the alpha subunit (alpha-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters . Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant . Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on alpha-CTD . Overall, our results suggest that CRP interaction with alpha-CTD may not be required for rhaBAD activation; however, alpha-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS. J Bacteriol, 2000 Jun, 182(12), 3467 - 74 Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA; Barbosa TM et al.; In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards . For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E . coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E . coli . Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression . Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups . Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon . Northern blot analysis of selected genes confirmed the results obtained with the macroarrays . The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described. J Bacteriol, 2000 Jun, 182(12), 3460 - 6 Analysis of guanine nucleotide binding and exchange kinetics of the Escherichia coli GTPase Era; Sullivan SM et al.; Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles . Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported . Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (N-methyl-3'-O-anthraniloyl) guanine nucleotide analogs . The equilibrium binding constants (K(D)) for mGDP and mGTP (0.61 +/- 0 . 12 microgM and 3.6 +/- 0.80 microM, respectively) are similar to those of the unmodified nucleotides . The single turnover rates for mGTP hydrolysis by Era were 3.1 +/- 0.2 mmol of mGTP hydrolyzed/min/mol in the presence of 5 mM MgCl(2) and 5.6 +/- 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl(2) . Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg(2+) . We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era. J Bacteriol, 2000 Jun, 182(12), 3377 - 82 A mutation in secY that causes enhanced SecA insertion and impaired late functions in protein translocation; Matsumoto G et al.; A cold-sensitive secY mutant (secY125) with an amino acid substitution in the first periplasmic domain causes in vivo retardation of protein export . Inverted membrane vesicles prepared from this mutant were as active as the wild-type membrane vesicles in translocation of a minute amount of radioactive preprotein . The mutant membrane also allowed enhanced insertion of SecA, and this SecA insertion was dependent on the SecD and SecF functions . These and other observations suggested that the early events in translocation, such as SecA-dependent insertion of the signal sequence region, is actually enhanced by the SecY125 alteration . In contrast, since the mutant membrane vesicles had decreased capacity to translocate chemical quantity of pro-OmpA and since they were readily inactivated by pretreatment of the vesicles under the conditions in which a pro-OmpA translocation intermediate once accumulated, the late translocation functions appear to be impaired . We conclude that this periplasmic secY mutation causes unbalanced early and late functions in translocation, compromising the translocase's ability to catalyze multiple rounds of reactions. J Bacteriol, 2000 Jun, 182(12), 3336 - 44 Cyanobacterial sulfide-quinone reductase: cloning and heterologous expression; Bronstein M et al.; The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus . SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively . Based on the conserved sequences, the gene encoding A . halophytica SQR was also cloned . The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O . limnetica SQR and 437 amino acids for A . halophytica SQR and show 58% identity and 74% similarity . The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O . limnetica SQR and A . halophytica SQR, respectively . A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans . All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints . The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter . Membranes isolated from E . coli cells expressing A . halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide . The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature. J Vet Med Sci, 2000 May, 62(5), 543 - 7 Lectin-binding capacity of glycoconjugates in Escherichia coli 09:K103:NM, 987P+ST+-infected porcine lower small intestines; Sohn YS et al.; Composition of glycoconjugates were investigated in Escherichia coli 09:K103:NM, 987P+ST+-infected lower small intestines of 1-week-old pigs by the use of twenty one biotinylated-labelled lectins with avidin-biotin-peroxidase complex method . Piglets with experimental group were inoculated by feeding 5 ml of culture inoculum (5 x 10(9) colony-forming units/ml) with 15 ml of milk replacer . At the onset of diarrhea, experimental piglets and time-matched control piglets were euthanatized using electrocution, necropsied, and tested by lectin histochemistry . As compared with control, staining intensity of seven lectins altered in ileal villus brush border and goblet cells of pigs inoculated with the pathogen. Zh Mikrobiol Epidemiol Immunobiol, 1999 Sep-Oct, (5), 109 - 12 {The determination of lipopolysaccharide admixtures in protein preparations}; Denisova LIa et al.; A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed . The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system . Proteins are previously removed from the solution by treatment with hot phenol . Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane. Zh Mikrobiol Epidemiol Immunobiol, 1999 May-Jun, (3), 41 - 6 {The antidiphtheria activity of the pre-EGF fragment}; Sergienko VI et al.; The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques . For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR) . After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen . This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208 . Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98% . Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein. J Biol Chem, 2000 Aug 25, 275(34), 25900 - 6 The human homolog of Escherichia coli Orn degrades small single-stranded RNA and DNA oligomers; Nguyen LH et al.; We report here the identification of human homologues to the essential Escherichia coli Orn protein and the related yeast mitochondrial DNA-escape pathway regulatory factor Ynt20 . The human proteins appear to arise from alternatively spliced transcripts, and are thus identical, except the human Ynt20 equivalent contains an NH(2)-terminal extension that possesses a predicted mitochondrial protease cleavage signal . In vitro analysis revealed that the smaller human protein exhibits a 3' to 5' exonuclease activity for small (primarily </=5 nucleotides in length) single-stranded RNA and DNA oligomers . We have named this human protein Sfn for small fragment nuclease to reflect its broad substrate range, and have termed the longer protein hSfnalpha . Sfn prefers Mn(2+) as a metal cofactor and displays a temperature-resistant (to 50 degrees C) nuclease activity . Kinetic analysis indicates that Sfn exhibits a similar affinity for small RNAs and DNAs (K(m) of approximately 1.5 micrometer), but degrades small RNAs approximately 4-fold more efficiently than DNA . Mutation of a conserved aspartate (Asp(136)) to alanine abolishes both nuclease activities of Sfn . Northern blot analysis revealed that a 1-kilobase transcript corresponding to SFN and/or SFNalpha (these mRNAs differ by only two nucleotides) is expressed at varying levels in all fetal and adult human tissues examined . Expressed tag sequence clone analysis found that the two splice variants, SFN to SFNalpha, are present at a ratio of roughly 4 to 1, respectively . The results presented within suggest a role for human Sfn in cellular nucleotide recycling. J Biol Chem, 2000 Aug 25, 275(34), 26467 - 76 Cleavage of holliday junctions by the Escherichia coli RuvABC complex; Eggleston AK et al.; The Escherichia coli RuvABC proteins process recombination intermediates during genetic recombination and recombinational repair . Although early biochemical studies indicated distinct RuvAB-mediated branch migration and RuvC-mediated Holliday junction resolution reactions, more recent studies have shown that the three proteins act together as a "resolvasome" complex . In this work we have used recombination intermediates made by RecA to determine whether the RuvAB proteins affect the sequence specificity of the RuvC resolvase . We find that RuvAB proteins do not alter significantly the site specificity of RuvC-dependent cleavage, although under certain conditions, they do affect the efficiency of cleavage at particular sites . The presence of RecA also influences cleavage at some sites . We also show that the RuvAB proteins act upon transient strand exchange intermediates made using substrates that have the opposite polarity of those preferred by RecA . Together, our results allow us to develop further a model for the recombinational repair of DNA lesions that lead to the formation of post-replication gaps during DNA replication . The novel features of this model are as follows: (i) the RuvABC resolvasome recognizes joints made by RecA; (ii) resolution by RuvABC occurs at specific sites containing the RuvC consensus cleavage sequence 5'-(A/T)TT downward arrow(G/C)-3'; and (iii) Holliday junction resolution often occurs close to the initiating gap without significant heteroduplex DNA formation. Curr Opin Biotechnol, 2000 Jun, 11(3), 244 - 9 Engineering dioxygenases for efficient degradation of environmental pollutants; Furukawa K; Dioxygenases have recently been engineered to improve their capabilities for environmental pollutant degradation . The techniques used to achieve this include in vitro DNA shuffling and subunit or domain exchanges between dioxygenases of different bacterial origins . Such evolved enzymes acquire novel and enhanced degradation capabilities of xenobiotic compounds, such as polychlorinated biphenyls, trichloroethylene and a variety of aromatic compounds . Hybrid strains in which the evolved genes are integrated into the chromosomal operons exhibit efficient degradation of xenobiotic chlorinated compounds. J Appl Physiol, 2000 Jun, 88(6), 1933 - 42 Correlation between ventilation and perfusion determines VA/Q heterogeneity in endotoxemia; Gerbino AJ et al.; Endotoxin increases ventilation-to-perfusion ratio (VA/Q) heterogeneity in the lung, but the precise changes in alveolar ventilation (VA) and perfusion that lead to VA/Q heterogeneity are unknown . The purpose of this study was to determine how endotoxin affects the distributions of ventilation and perfusion and the impact of these changes on VA/Q heterogeneity . Seven anesthetized, mechanically ventilated juvenile pigs were given E . coli endotoxin intravenously, and regional ventilation and perfusion were measured simultaneously by using aerosolized and injected fluorescent microspheres . Endotoxemia significantly decreased the correlation between regional ventilation and perfusion, increased perfusion heterogeneity, and redistributed perfusion between lung regions . In contrast, ventilation heterogeneity did not change, and redistribution of ventilation was modest . The decrease in correlation between regional ventilation and perfusion was responsible for significantly more VA/Q heterogeneity than were changes in ventilation or perfusion heterogeneity . We conclude that VA/Q heterogeneity increases during endotoxemia primarily as a result of the decrease in correlation between regional ventilation and perfusion, which is in turn determined primarily by changes in perfusion. Arch Biochem Biophys, 2000 May 15, 377(2), 366 - 71 Limited proteolysis of branching enzyme from Escherichia coli; Binderup K et al.; Branching enzyme is involved in determining the structure of starch and glycogen . It catalyzes the formation of branch points by cleavage and transfer of alpha-1,4-glucan chains to alpha-1,6 branch points . Branching enzyme belongs to the amylolytic family of enzymes containing four conserved regions in a central (alpha/beta)8-barrel . Limited proteolysis of the branching enzyme from Escherichia coli (84 kDa) by proteinase K produced a truncated protein of 70-kDa, which still retained 40-60% of branching activity, depending on the type of assay used . Amino acid sequencing showed that the 70-kDa protein lacked 111 or 113 residues at the amino terminal, whereas the carboxy terminal was still intact . We purified this truncated enzyme to homogeneity and analyzed its properties . The enzyme had a three- to fourfold lower catalytic efficiency than the native enzyme, whereas the substrate specificity was unaltered . Furthermore, a branching enzyme with 112 residues deleted at the amino terminal was constructed by recombinant technology and found to have properties identical to those of the proteolyzed enzyme. Invest Ophthalmol Vis Sci, 2000 Jun, 41(7), 1812 - 7 Reduced leukocyte migration, but normal rolling and arrest, in interleukin-8 receptor homologue knockout mice; Becker MD et al.; PURPOSE: To determine the role of the murine interleukin-8 receptor homologue (mIL-8Rh, neutrophil chemokine CXC receptor 2) in leukocyte migration using intravital microscopy in a standardized model of eye inflammation, endotoxin-induced uveitis (EIU) . METHODS: Two hundred fifty nanograms of E . coli endotoxin was injected into the vitreous of knockout mIL-8Rh(-/-) (n = 7) mice or heterozygous littermate mIL-8Rh(+/-) controls (n = 7) . Intravital microscopic examination of iris microvasculature was performed at baseline and 6 and 24 hours after endotoxin injection . The numbers of rolling (cells/mm2 endothelial surface/min), sticking (cells/mm2 endothelial surface), and infiltrating cells (cells/mm2 iris tissue) were evaluated by digital off-line quantification . RESULTS: The number of infiltrating cells was significantly reduced in mIL-8Rh(-/-) mice: 406 +/- 77 cells/mm2 at 6 hours and 242 +/- 50 cells/mm2 at 24 hours in mIL-8Rh(+/-) mice versus 14 +/- 4 cells/mm2 at 6 hours and 38 +/- 11 cells/mm2 at 24 hours in mIL-8Rh(-/-) mice (P < 0.001) . In contrast, the absence of the IL-8 receptor homologue did not reduce rolling or sticking . CONCLUSIONS: Iris rhodamine angiography allows precise quantification of leukocyte-endothelial dynamics in the absence of surgical trauma . IL-8 and its homologues are known to be potent signals for leukocyte migration . Although IL-8 has previously been implicated in cell adhesion, video imaging in vivo demonstrated that deletion of the IL-8 receptor homologue had minimal effect on rolling or arrest in this model of inflammation. Plant Cell Physiol, 2000 Apr, 41(4), 495 - 502 Molecular and biochemical characterization of a novel hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6"-O-acyltransferase from Perilla frutescens; Yonekura-Sakakibara K et al.; We have isolated and characterized a cDNA, PfAT208, encoding hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6"-O-acyltransferase (3AT) from Perilla frutescens . The identity of the cDNA was established by determination of the reaction products with recombinant enzyme overexpressed in Escherichia coli . The deduced amino acid sequence has a few regions that are conserved in a CoA-dependent acyltransferase family . The recombinant enzyme produced in yeast could utilize cyanidin 3-glucoside and cyanidin 3,5-diglucoside, putative substrates in vivo, as well as other anthocyanins . The inhibitory effects of diethyl pyrocarbonate and N-ethylmaleimide on the recombinant 3AT activities suggest that histidine and cysteine residues are important for their catalytic function . These properties are in common with anthocyanin 5-O-glucoside-6"-O-acyltransferase (5AT) . In Northern analysis, a transcript of PfAT208 was detected in the young leaves of perilla red forma . The properties of other cDNAs, gentian GAT106 and petunia PhAT48, isolated during the above cloning procedure are also described. Plant Cell Physiol, 2000 Apr, 41(4), 465 - 76 Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase; Yamaguchi Y et al.; Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively . Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A . thaliana genome . Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E . coli clearly showed cysteine synthase activity . In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not . Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo . The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A . thaliana seedlings were subjected to various stresses, including nutrient deprivation . In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme. Mutat Res, 2000 May 31, 459(4), 275 - 84 Enhanced Tn10 and mini-Tn10 precise excision in DNA replication mutants of Escherichia coli K12; Nagel R et al.; The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli . dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III . Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements . The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal . The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations . A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results. Eur J Pharmacol, 2000 Jun 2, 397(2-3), R3 - 5 Altered behaviour following RNA interference knockdown of a C . elegans G-protein coupled receptor by ingested double stranded RNA; Vaz Gomes A et al.; Using a systemic and continuous delivery method based on feeding on a particular strain of transformed Escherichia coli to induce double stranded RNA-mediated interference, we targeted the product of the npr-1 gene, a putative Caenorhabditis elegans homologue of a neuropeptide Y receptor, a G-protein coupled receptor . We were able to reproduce the social behaviour observed for the naturally occurring npr-1 mutant when wild type N2 Bristol eggs developed in a lawn of bacteria producing double stranded RNA for npr-1 . This facile approach may also be useful when studying the function of other worm G-protein coupled receptors. J Biol Chem, 2000 Sep 22, 275(38), 29200 - 6 Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein; Merkel JS et al.; We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel . After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis . Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence . Most of the fluorescent pairs have predominantly charged and polar guest site residues . The magnitude and the rate of fluorescence acquisition in vivo from transformed E . coli cells varies among the mutants despite comparable levels of protein expression . Spectroscopic measurements of purified mutants show that the native protein structure is maintained . Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates . Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding . Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments. J Biol Chem, 2000 Aug 18, 275(33), 25299 - 307 The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins; Zhang B et al.; The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells . Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA . Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration . The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides . The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins . The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism . Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present . Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor . These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases . The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity. J Mol Biol, 2000 Jun 16, 299(4), 1147 - 54 Conservation of substructures in proteins: interfaces of secondary structural elements in proteasomal subunits; Gille C et al.; It is observed that during divergent evolution of two proteins with a common phylogenetic origin, the structural similarity of their backbones is often preserved even when the sequence similarity between them decreases to a virtually undetectable level . Here we analyzed, whether the conservation of structure along evolution involves also the local atomic structures in the interfaces between secondary structural elements . We have used as study case one protein family, the proteasomal subunits, for which 17 crystal structures are known . These include 14 different subunits of Saccharomyces cerevisiae, 2 subunits of Thermoplasma acidophilum and one subunit of Escherichia coli . The structural core of the 17 proteasomal subunits has 23 secondary structural elements . Any two adjacent secondary structural elements form a molecular interface consisting of two molecular patches . We found 61 interfaces that occurred in all 17 subunits . The 3D shape of equivalent molecular patches from different proteasomal subunits were compared by superposition . Our results demonstrate that pairs of equivalent molecular patches show an RMSD which is lower than that of randomly chosen patches from unrelated proteins . This is true even when patch comparisons with identical residues were excluded from the analysis . Furthermore it is known that the sequential dissimilarity is correlated to the RMSD between the backbones of the members of protein families . The question arises whether this is also true for local atomic structures . The results show that the correlation of individual patch RMSD values and local sequence dissimilarities is low and has a wide range from 0 to 0.41, however, it is surprising that there is a good correlation between the average RMSD of all corresponding patches and the global sequence dissimilarity . This average patch RMSD correlates slightly stronger than the C(alpha)-trace RMSD to the global sequence dissimilarity . J Mol Biol, 2000 Jun 16, 299(4), 993 - 1003 Compromise and accommodation in ecotin, a dimeric macromolecular inhibitor of serine proteases; Gillmor SA et al.; Ecotin is a dimeric serine protease inhibitor from Escherichia coli which binds proteases to form a hetero-tetramer with three distinct interfaces: an ecotin-ecotin dimer interface, a larger primary ecotin-protease interface, and a smaller secondary ecotin-protease interface . The contributions of these interfaces to binding and inhibition are unequal . To investigate the contribution and adaptability of each interface, we have solved the structure of two mutant ecotin-trypsin complexes and compared them to the structure of the previously determined wild-type ecotin-trypsin complex . Wild-type ecotin has an affinity of 1 nM for trypsin, while the optimized mutant, ecotin Y69F, D70P, which was found using phage display technologies, inhibits rat trypsin with a K(i) value of 0.08 nM . Ecotin 67-70A, M84R which has four alanine substitutions in the ecotin-trypsin secondary binding site, along with the M84R mutation at the primary site, has a K(i) value against rat trypsin of 0.2 nM . The structure of the ecotin Y69F, D70P-trypsin complex shows minor structural changes in the ecotin-trypsin tetramer . The structure of the ecotin 67-70A, M84R mutant bound to trypsin shows large deviations in the tertiary and quaternary structure of the complex . The trypsin structure shows no significant changes, but the conformation of several loop regions of ecotin are altered, resulting in the secondary site releasing its hold on trypsin . The structure of several regions previously considered to be rigid is also significantly modified . The inherent flexibility of ecotin allows it to accommodate these mutations and still maintain tight binding through the compromises of the protein-protein interfaces in the ecotin-trypsin tetramer . A comparison with two recently described ecotin-like genes from other bacteria suggests that these structural and functional features are conserved in otherwise distant bacterial lineages . J Mol Biol, 2000 Jun 16, 299(4), 979 - 91 Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals; Fresquet V et al.; Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine . The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain . Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP . Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation . S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays . V994A also decreased significantly IMP and UMP binding . L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide . K993W, R1020A, R1021A and K1061A were without substantial effects . The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex . They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation . K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding . These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain . J Mol Biol, 2000 Jun 16, 299(4), 941 - 51 Differential role of the intermolecular base-pairs G292-C(75) and G293-C(74) in the reaction catalyzed by Escherichia coli RNase P RNA; Busch S et al.; We present a systematic investigation of the thermodynamic and kinetic role of the intermolecular G292-C(75 )and G293-C(74 )Watson-Crick base-pairs in the reaction catalyzed by Escherichia coli RNase P RNA . Single turnover kinetics were analyzed for wild-type RNase P RNA and two variants with a single G to C exchange (C292 or C293), either acting on wild-type precursor tRNA (ptRNA) or derivatives carrying a complementary change at the tRNA 3'-end (G(74)CA or CG(75)A) . Ground state binding of tRNA was studied using three different methods, including a novel fluorescence-based assay measuring equilibrium binding . We conclude that: (1) the role of the G293-C(74 )interaction is essentially confined to Watson-Crick base-pairing, with no indication for crucial tertiary contacts involving this base-pair; (2) the G293-C(74 )pair, although being as important for ptRNA ground state binding as G292-C(75), is much less crucial to catalytic performance than the G292-C(75) pair; (3) disruption of the G292-C(75 )base-pair results in preferential destabilization of enzyme transition-state complexes; and (4) the identity of the G292-C(75) pair, as part of the higher-order structural context consisting of coplanar G292-C(75)-A258 and G291-G259-A(76 )triples, contributes to high affinity binding of ptRNA and catalytic efficiency . J Mol Biol, 2000 Jun 16, 299(4), 897 - 905 Estimating the number of protein folds and families from complete genome data; Wolf YI et al.; Using the data on proteins encoded in complete genomes, combined with a rigorous theory of the sampling process, we estimate the total number of protein folds and families, as well as the number of folds and families in each genome . The total number of folds in globular, water- soluble proteins is estimated at about 1000, with structural information currently available for about one-third of the number . The sequenced genomes of unicellular organisms encode from approximately 25%, for the minimal genomes of the Mycoplasmas, to 70-80% for larger genomes, such as Escherichia coli and yeast, of the total number of folds . The number of protein families with significant sequence conservation was estimated to be between 4000 and 7000, with structures available for about 20% of these . J Mol Biol, 2000 Jun 16, 299(4), 865 - 74 Length of CTG.CAG repeats determines the influence of mismatch repair on genetic instability; Parniewski P et al.; We showed previously that mutations in methyl-directed mismatch repair of Escherichia coli reduced the occurrence of large deletions in (CTG.CAG)(175) repeats contained on plasmids . By contrast, other workers reported that mutations in mismatch repair increase the frequency of small-length changes in the shorter (CTG.CAG)(64) . Using plasmids with a variety of lengths and purity of (CTG.CAG) repeats, we have resolved these apparently conflicting observations . We show that all lengths of (CTG.CAG) repeats are subject to small-length changes (<eight repeats) upon inactivation of the mismatch repair pathway . However, large deletions (>eight repeats) in (CTG.CAG)(n) occur more readily in cells with active mismatch repair . The frequency of large deletions is proportional to the tract length; in our assays they become prominent in tracts greater than 100 repeats . Interruptions in repeat purity enhance the occurrence of large deletions . In addition, we observed a high level of incidence of deletions in (CTG.CAG) repeats for cultures passing repeatedly through stationary phase during long-term growth experiments of all strains (i.e . with active or inactive mismatch repair) . These results agree with current theories on mismatch repair acting on DNA slippage events that occur in DNA triplet-repeats . Cytokine, 2000 Jun, 12(6), 822 - 7 Inflammatory and immunological parameters in children with haemolytic uremic syndrome (HUS) and gastroenteritis-pathophysiological and diagnostic clues; Westerholt S et al.; The objective of this study was to identify parameters indicating a risk for developing typical haemolytic uremic syndrome (D+HUS) during the prodromal phase of diarrhea caused by enterohaemorrhagic Escherichia coli (EHEC) . Forty-eight children were studied prospectively with regard to inflammatory serum factors on admission to hospital . Ten patients developed D+HUS (group I), 15 suffered from viral-gastroenteritis (group IIa) and 23 from other types of bacterial gastroenteritis (group IIb) . Mean levels of IL-8 tended to be elevated in group I compared to groups IIa and IIb . Neopterin and IL-10 levels particularly were significantly decreased in HUS in comparison to both gastroenteritis groups . Low IL-10 levels indicate a substantial disregulation of the immune response in HUS, as IL-10 downregulates the pro-inflammatory response and suppresses pro-coagulant activity in experimental endotoxemia . Our results suggest low neopterin, high IL-8 and especially low IL-10 levels are indicators of a high risk for developing HUS . Cytokine, 2000 Jun, 12(6), 786 - 90 Targeting activated lymphocytes with an entirely human immunotoxin analogue: human pancreatic RNase1-human IL-2 fusion; Psarras K et al.; A hybrid human protein was produced in E . coli by fusing the genes encoding human pancreatic RNase1 (hpRNase1) and human IL-2 (hIL-2) . The recombinant hpRNase1-hIL-2 inhibited protein synthesis in HTLV-1-infected, malignant T cells, which hyperproduce high affinity IL-2 receptors, with an IC(50)of 2x10(-8) M, whereas no inhibition was detectable in control cells with lower affinity receptors . HpRNase1 alone had an IC(50)of almost 10(-3) M . A molar excess of hIL-2 blocked the protein synthesis inhibition dose-dependently . In a human mixed lymphocyte culture, hpRNase1-hIL-2 inhibited the proliferation of responder cells with potency comparable to that of cyclosporine, while non-effective doses of FK506 importantly improved its potency . Despite its short half-life in animals, hpRNase1-hIL-2 rapidly enters cells in a few minutes and arrests the protein translation in less than 10 h . Thus, hpRNase1-hIL-2 may be useful to selectively eliminate activated lymphocytes hyperproducing high affinity IL-2 receptors, as in allograft rejection, graft-versus-host disease, autoimmune disorders, adult T cell leukaemia and other lymphoproliferative or retroviral malignancies including HIV infection, without inducing general immunosuppression . As an entirely human "immunotoxin analogue" it may alleviate the dose limiting toxicity and immunogenicity of conventional immunotoxins . Cytokine, 2000 Jun, 12(6), 573 - 7 Expression and purification of woodchuck tumour necrosis factor alpha; Lohrengel B et al.; The production of recombinant woodchuck cytokines is an essential prerequisite to study the immune response to hepadnavirus infection in the woodchuck model . Woodchuck tumour necrosis factor-alpha (TNF-alpha) was expressed in mammalian cells and in Escherichia coli . A test system for the biological activity of woodchuck TNF-alpha was established on basis of its cytotoxic effect to the murine fibrosarcoma cell line L929 . Recombinant TNF-alpha was purified and used for the production of neutralizing antisera . Biochemistry, 2000 Jun 20, 39(24), 7316 - 9 Role of an interdomain Gly-Gly sequence at the regulatory-substrate domain interface in the regulation of Escherichia coli . D-3-phosphoglycerate dehydrogenase; Grant GA et al.; The regulatory and substrate binding domains of D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) from Escherichia coli are connected by a single polypeptide strand that contains a Gly-Gly sequence approximately midway between the domains . The potential flexibility of this sequence and its strategic location between major domain structures suggests that it may function in the conformational change leading from effector binding to inhibition of the active site . Site-directed mutagenesis of this region (Gly-336-Gly-337) supports this hypothesis . When bulky side chains were substituted for the glycines at these positions, substantial changes in the ability of serine to inhibit the enzyme were seen with little effect on the activity of the enzyme . The effect of these substitutions could be alleviated by placing a new glycine residue at position 335, immediately flanking the original glycine pair . On the other hand, substituting a glycine at position 338 revealed a critical role for the side chain of Arg-338 . This residue may function in stabilizing the conformation about the Gly-Gly turn, resulting in a specific orientation of the adjacent domains relative to each other . Rotation about the phi or psi bonds of either Gly-336 or Gly-337 would have a profound effect on this orientation . The data are consistent with this as a role for the Gly-Gly sequence between the regulatory and substrate binding domains of PGDH. Biochemistry, 2000 Jun 20, 39(24), 7309 - 15 15N isotope effects in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase: evidence for a tetrahedral intermediate in the mechanism; Rishavy MA et al.; 15N isotope effects have been measured on the hydrolysis of glutamine catalyzed by carbamyl phosphate synthetase of Escherichia coli . The isotope effect in the amide nitrogen of glutamine is 1 . 0217 at 37 degrees C with the wild-type enzyme in the presence of MgATP and HCO(3)(-) (overall reaction taking place) . This V/K isotope effect indicates that breakdown of the tetrahedral intermediate formed with Cys 269 to release ammonia is the rate-limiting step in the hydrolysis . A full isotope effect of 1 . 0215 is also seen in the partial reaction catalyzed by an E841K mutant enzyme, whose rate of glutamine hydrolysis is not affected by MgATP and HCO(3)(-) . With wild-type enzyme in the absence of MgATP and HCO(3)(-), however, the (15)N isotope effect is reduced to 1 . 0157 . These isotope effects are interpreted in terms of partitioning of the tetrahedral intermediate whose rate of formation is dependent upon a conformation change which closes the active site after glutamine binding and prepares the enzyme for catalysis . An Ordered Uni Bi mechanism for glutamine hydrolysis that is consistent with the isotope effects and with the catalytic properties of the enzyme is proposed. Biochemistry, 2000 Jun 20, 39(24), 7300 - 8 Intersubunit association induces unique allosteric dependence of the T127L CRP mutant on pH; Shi Y et al.; The allosteric activation of the T127-->L mutant of 3',5'-cyclic adenosine monophosphate (cAMP) receptor protein (CRP) by cAMP changes from an exothermic, independent two-site binding mechanism at pH 7.0 to an endothermic, interacting two-site binding mechanism at pH 5.2, similar to that observed for CRP at pH 7.0 and 5.2 . Since the T127-->L mutation at the subunit interface of the CRP dimer creates a more perfect leucine-zipper motif, it is believed to increase the intersubunit association and the stability of the CRP, as is observed by the higher thermal stability of the T127L mutant relative to that of CRP in differential scanning calorimetry (DSC) measurements . The DSC scans also exhibit a single thermal denaturation transition for CRP and a S128A mutant from pH 5.2 to 7 . 0, while the broader transition peak of the T127L mutant becomes resolvable into two transitions below pH < or =5.2 . Circular dichroism measurements on T127L and CRP at pH 7.0 and 5.2 show changes in the tertiary structure of both proteins with the exception of the tertiary structure around the two tryptophan residues in the amino-terminal domain . Although gel electrophoresis of the proteolysis (pH 5.2) products of T127L, CRP, and their cAMP- and cGMP-ligated complexes shows the subunit band and an amino-terminal domain fragment band, the fully allosterically activated complexes of T127L and CRP show the amino-terminal domain fragment band but not the subunit band . The results are interpreted in terms of the allosteric activation of CRP by cAMP by a conformational change from an "open" to a "closed" form of CRP, which involves changes in the tertiary structure of the carboxyl-terminal domains that are partially induced by an increase in the intersubunit association in T127L . While T127L retains its intersubunit association from pH 5.2 to 7.0, changes occur in the carboxyl-terminal domain so that the endothermic, allosteric activation mechanism of CRP by cAMP is restored in T127L at pH 5.2. Biochemistry, 2000 Jun 20, 39(24), 7276 - 83 Mutational evidence of transition state stabilization by serine 88 in Escherichia coli type I signal peptidase; Carlos JL et al.; Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins . SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mechanism instead of the classical Ser/His/Asp triad found in most Ser proteases . Recent X-ray crystallographic studies indicate that the backbone amide nitrogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole . In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants . The S88T mutant maintains near-wild-type activity with the substrate pro-OmpA nuclease A . In contrast, substitution with Cys at position 88 results in more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease) . Measurements of the kinetic constants of the individual mutant enzymes indicate that these decreases in activity are attributed mainly to decreases in k(cat) while effects on K(m) are minimal . Thermal inactivation and CD spectroscopic analyses indicate no global conformational perturbations of the Ser 88 mutants relative to the wild-type E . coli SPase I enzyme . These results provide strong evidence for the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E . coli SPase I. Biochemistry, 2000 Jun 20, 39(24), 7063 - 73 NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha; Moy FJ et al.; Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis . The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy . A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints . The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0 . 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains . The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1 . 94 A between the free and bound forms of RGS4 . The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site . A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices . The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1) . The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(al