Escherichia coli

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)(alpha)(1) X-ray structure . This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1) . The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.

Biochemistry (Mosc), 2000 May, 65(5), 546 - 53
Human recombinant laminin-binding protein: isolation, purification, and crystallization; Sorokin AV et al.; The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned . The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells . The human LBP was expressed by E . coli cells, and it was purified using Ni-NTA-Sepharose chromatography . The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD . A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot . The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP . Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus . The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein . For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique . The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size . The X-ray diffraction field of the crystal extended to 2.5 A.

J Bacteriol, 2000 Jul, 182(13), 3858 - 62
Phosphorelay as the sole physiological route of signal transmission by the arc two-component system of Escherichia coli; Kwon O et al.; The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions . In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutant arcB alleles . The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.

J Bacteriol, 2000 Jul, 182(13), 3802 - 8
Fur positive regulation of iron superoxide dismutase in Escherichia coli: functional analysis of the sodB promoter; Dubrac S et al.; In Escherichia coli, the expression of sodB, which encodes iron superoxide dismutase, has been suggested to be activated by Fur, the iron-responsive global regulator initially characterized as a transcriptional repressor . We investigated sodB regulation by functional analysis of the sodB promoter using sodB-lac fusions with various truncated and mutated promoters . Several cis- and trans-acting elements involved in sodB regulation have been identified . The beta-galactosidase activity of sodB-lacZ reporter fusions and RNA analysis showed sevenfold iron-dependent, Fur-mediated activation of expression . A region just downstream from -10, including a large palindromic sequence encompassing the +1 position followed by a 14-bp AT-rich motif, is the site of Fur positive regulation, and the integrity of both sequences was required for full Fur-mediated activation . The life span of sodB mRNA was three times longer in a fur(+) strain, indicating that Fur-mediated activation proceeds, at least in part, at the posttranscriptional level . The H-NS and IHF histone-like factors also affected sodB expression . IHF slightly repressed sodB expression independently of Fur regulation . In contrast, H-NS negative regulation operated only in the absence of Fur . Remarkably, psodB behaved like a "pure extended -10" promoter . Deletion of the -35 region did not affect expression, whereas expression was totally abolished by a TG-to-CC mutation in the extended -10 sequence TGcTACCCT.

J Bacteriol, 2000 Jul, 182(13), 3734 - 9
Temperature-dependent function of the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel and coupling with glycinamide ribonucleotide synthetase in a hyperthermophile; Bera AK et al.; Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity . Both enzymes were maximally active at a temperature of at least 90 degrees C, with half-lives of 65 min for GPAT and 60 h for GARS at 80 degrees C . GPAT activity is known to depend upon channeling of NH(3) from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place . The efficiency of channeling of NH(3) for synthesis of PRA was found to increase from 34% at 37 degrees C to a maximum of 84% at 80 degrees C . The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90 degrees C . Evidence was obtained for coupling between GPAT and GARS for PRA transfer . The coupling was temperature dependent, exhibiting a transition between 37 and 50 degrees C, and remained relatively constant up to 90 degrees C . The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range . These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.

J Bacteriol, 2000 Jul, 182(13), 3688 - 92
Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea; Graupner M et al.; Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococcus jannaschii and one from Methanothermus fervidus were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following alpha-hydroxy-alpha-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-alpha-hydroxyglutaric acid and alpha-ketoglutaric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4, 6-hexanetetracarboxylic acid . Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F(420), and methanofuran, respectively . Both the MJ1425-encoded enzyme and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (S)-malate using either NADH or NADPH as a reductant . Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (S)-sulfolactate, but the V(max)/K(m) value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nucleotides . Both the M . fervidus and the MJ1425-encoded enzyme catalyzed the NAD(+)-dependent oxidation of (S)-sulfolactate to sulfopyruvate . The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of alpha-ketoglutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin . Neither of the enzymes reduced pyruvate to (S)-lactate, a component of coenzyme F(420) . Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that is not involved in the biosynthesis of methanofuran c . We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.

J Bacteriol, 2000 Jul, 182(13), 3626 - 31
Characterization of a bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase; Fuchs TM et al.; The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs) . In line with the successful complementation of the E . coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E . coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively . The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5'-phosphate binding motif . Enzymatic studies revealed that this ORF encodes a protein with N-succinyl-L,L-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-L,L-DAP, the substrate of the desuccinylase DapE . Therefore, this gene appears to encode the DapC protein of B . pertussis . Apart from the pyridoxal-5'-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-L,L-DAP aminotransferase activity, ArgD of E . coli.

J Enzyme Inhib, 2000, 15(1), 1 - 10
A kinetic study on the interaction between tazobactam (a penicillanic acid sulphone derivative) and active-site serine beta-lactamases; Perilli M et al.; The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry . Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam . A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.

JPEN J Parenter Enteral Nutr, 2000 May-Jun, 24(3), 159 - 63
Fish oil modulates macrophage P44/P42 mitogen-activated protein kinase activity induced by lipopolysaccharide; Lo CJ et al.; BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades represent a major signal system to transduce extracellular signals into cellular responses . Overactivity of MAPK has been implicated in the development of many diseases, including cancer and sepsis . This study investigated the hypothesis that fish oil altered the membrane phospholipid composition and modulated MAPK activity . METHODS: RAW 264.7 cells, a mouse macrophage (Mphi) cell line, were grown in eicosapentaenoic acid (EPA)-rich media (114 micromol/L) for 48 hours . Mphi were washed and exposed to Escherichia coli lipopolysaccharide (LPS; 1 microg/mL) for 10 minutes . Both total and activated (phosphorylated) portions of MAPK (P44 and P42) were determined by Western blot assays . AP-1 transcription factor activity was determined by electrophoretic mobility gel shift assays (EMSA) . Mphi tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays . RESULTS: LPS stimulation induced RAW cell phosphorylation of P44/P42 . In contrast, RAW cells grown in EPA-rich media had less P44/P42 activation in the presence of LPS . Total P44/P42 were not affected by EPA or LPS . Similarly, EPA also inhibited AP-1 activity . Inhibition of P44/P42 activity with PD98059 reduced both AP-1 activity and TNF mRNA expression of LPS-stimulated Mphi . CONCLUSIONS: Our data suggest that fish oil regulates macrophage proinflammatory gene activation, at least in part, by modulating the MAPK activity.

Protein Sci, 2000 May, 9(5), 1038 - 41
NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori; Damberger F et al.; NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0 . At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0 . Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4 . The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion . Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.

Protein Sci, 2000 May, 9(5), 1035 - 7
Temperature-sensitive suppressor mutations of the Escherichia coli DNA gyrase B protein; Blance SJ et al.; Escherichia coli strain LE316 contains a mutation in gyrB that results in the substitution of Val164 to Gly and confers both chlorobiocin resistance and temperature sensitivity . Selection for suppressors of the ts phenotype yielded second-site mutations in GyrB at His38 and Thr157 . The properties of proteins bearing these mutations have been characterized, and a mechanism of suppression is proposed based upon structural considerations.

Protein Sci, 2000 May, 9(5), 991 - 1001
Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat); Cao QN et al.; The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined . The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase . This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var . awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2 . After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000 . Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin . Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215 . Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala . Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added . The results show that the putative hydrogen bond alone is responsible for the increase . We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Protein Sci, 2000 May, 9(5), 964 - 75
Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: studies of thrombic fragments of calmodulin; Brokx RD et al.; Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments . TM1 (1-106) and TM2 (107-148) . NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix . Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone . This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex . Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine . However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes . This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage . The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.

Protein Sci, 2000 May, 9(5), 907 - 15
Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes; Holtz KM et al.; Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported . The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively . The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively . Refinement of the AP-inhibitor complexes was carried out using X-PLOR . The final round of refinement was done using SHELXL-97 . Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds . The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures . The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate . In contrast, MMP binds with its phosphonate moiety directed toward solvent . Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H...X.

Nature, 2000 Jun 1, 405(6786), 590 - 3
Engineering stability in gene networks by autoregulation; Becskei A et al.; The genetic and biochemical networks which underlie such things as homeostasis in metabolism and the developmental programs of living cells, must withstand considerable variations and random perturbations of biochemical parameters . These occur as transient changes in, for example, transcription, translation, and RNA and protein degradation . The intensity and duration of these perturbations differ between cells in a population . The unique state of cells, and thus the diversity in a population, is owing to the different environmental stimuli the individual cells experience and the inherent stochastic nature of biochemical processes (for example, refs 5 and 6) . It has been proposed, but not demonstrated, that autoregulatory, negative feedback loops in gene circuits provide stability, thereby limiting the range over which the concentrations of network components fluctuate . Here we have designed and constructed simple gene circuits consisting of a regulator and transcriptional repressor modules in Escherichia coli and we show the gain of stability produced by negative feedback.

Nature, 2000 Jun 1, 405(6786), 586 - 90
Intraprotein radical transfer during photoactivation of DNA photolyase; Aubert C et al.; Amino-acid radicals play key roles in many enzymatic reactions . Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine . It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons . The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer . Available experimental data do not clearly discriminate between these proposals . We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy . Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps . After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation . A similar process may take place in photolyase-like blue-light receptors.

Nature, 2000 Jun 1, 405(6786), 537 - 43
Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand; Boyington JC et al.; Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules . Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide . KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide . No significant conformational changes in KIR occur on complex formation . The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor . Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding . Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity . KIR contact requires position 8 of the peptide to be a residue smaller than valine . A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Mol Cells, 2000 Apr 30, 10(2), 236 - 40
A novel technique for the effective production of short peptide analogs from concatameric short peptide multimers; Lee SJ et al.; We designed a basic unit of the modified chicken gonadotropin releasing hormone II (cGnRH-II) peptide containing a trypsin cleavable linker peptide at both ends of the original peptide . We made a synthetic DNA coding for the modified cGnRH-II peptide with asymmetric and complementary cohesive ends of linker nucleotides . A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units . The expressed peptide multimers were purified and subject to amino-terminal sequence analysis, which displayed the amino acid sequences expected from the designed nucleotides of the expression cassette . The monomeric cGnRH-II peptide analogs were generated after trypsin digestion . The present results showed that the technique developed for the production of the concatameric peptide multimers with cleavable linker peptides can be generally applicable to the production of short peptide analogs.

Mol Cells, 2000 Apr 30, 10(2), 220 - 5
Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy; Back K et al.; Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum . This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907 . The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe . The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities . The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.

Mol Cells, 2000 Apr 30, 10(2), 186 - 92
Immunological detection of serpin in the fall webworm, Hyphantria cunea and its inhibitory activity on the prophenoloxidase system; Park DS et al.; We previously identified a serine type protease inhibitor (serpin) cDNA, using PCR-based differential display, in the fall webworm which was up-regulated following a bacterial challenge (Shin et al., 1998) . The serpin cDNA was inserted into an expression vector and the serpin protein was expressed in Escherichia coli . In order to investigate the action of serpin in vivo, we examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabbit injected with recombinant serpin . H . cunea serpin was found mainly in the plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis . The concentration of serpin in the plasma was slightly increased following bacterial challenge . A new 50.5 kDa (approx.) band was detected post E . coli and distilled water injection . Both E . coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma, although E . coli injection produced a larger increase in activity . Hyphantria serpin probably participates in negative regulation of the prophenoloxidase (proPO) cascade . Recombinant serpin inhibits PO activity in the hemocyte lysate fraction activated by LPS . There is a similarity between the P2-P2' region (NKFG) of the serpin reactive site loop and the S2-S2' region (NRFG) of the insect proPO maturation site . This indicates a form of competitive inhibition of serpin against a protease involved in the activation of proPO . A tyrosine residue in the P11 region of serpin, which is conserved in the S11 regions of all known proPOs maturation sites, provides further support for this hypothesis.

Mol Cells, 2000 Apr 30, 10(2), 148 - 55
Molecular cloning, expression, and purification of nuclear inclusion A protease from tobacco vein mottling virus; Hwang DC et al.; The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue . The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography . The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively . The Km and k(cat) values were about 0.2 mM and 0.071 s(-1), respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease . In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases . These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.

Mol Cells, 2000 Apr 30, 10(2), 135 - 41
Isolation and characterization of cDNAs encoding ribosome inactivating protein from Dianthus sinensis L; Cho HJ et al.; To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity . Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa . All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70-90% inhibition of viral infectivity . In an attempt to search for the RIP gene from D . sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)+ RNA from D . sinensis leaves . DNA gel blot analysis showed that D . sinensis has multi-copy RIP genes . The expression of RIP gene was investigated in the flower, leaf, root and stem of D . sinensis, and was found to be most abundant in the leaf . Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D . sinensis leaves . They were divided into three groups on the basis of their nucleotide sequence homology . The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced . They all had an open reading frame of 882 bp . The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes . From the analysis of deduced amino acid sequences, it was predicted that D . sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus . When the cDNA was expressed in E . coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E . coli cells.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 1039 - 44
Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate; Miyake M et al.; Phosphotransacetylase (Pta) catalyzes the reversible conversion of acetyl-coenzyme A (CoA) to acetyl phosphate . Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha . During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type . PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio) . The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation . These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta . A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 447 - 53
Salinity-regulated replication of the endogenous plasmid pSY10 from the marine cyanobacterium Synechococcus sp; Takeyama H et al.; The endogenous plasmid pSY10 in the marine cyanobacterium Synechococcus sp . NKBG042902 is maintained at a high copy number when cells are grown in seawater and at a low copy number when cultured in freshwater . The mechanism of salinity-regulated replication of this plasmid was investigated . Transcription of repA was depressed under freshwater, which was accompanied by a low copy number of pSY10 and the appearance of a new protein that was expressed only in cells cultured in freshwater . This protein was observed to bind to putative repA promoters (Prep1 and Prep2) on pSY10 . Moreover, this protein was observed only in Synechococcus sp . NKBG042902 . The data suggest that this protein(s) regulates repA transcription in pSY10, stress responsive and encoded by the host chromosome.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 381 - 90
Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient; Hong K et al.; Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia eutropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported . The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal sigma 70 promotor . The pKS/PHB was the most efficient plasmid for phb operon expression among the three plasmids used: i.e., pKS-, pAED4, and pJM9131 . It was observed that isopropyl-beta-D-thiogalactopyranoside was not required for the induction of the expression of phb operon . The cell dry wt and polyhydroxyalkanoate content by E . coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.

J Biol Chem, 2000 Jun 16, 275(24), 18574 - 80
ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3; Shi Z et al.; Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions . Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability . IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified . We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins . A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3 . This interaction involved the cysteine-rich domain of ADAM 12 . Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein . To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells . Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12 . Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells . Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3 . The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed . The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.

J Biol Chem, 2000 Jun 16, 275(24), 18382 - 90
Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase; Hartenstine MJ et al.; Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange . Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases . Using self-priming sequences (e.g . (CAG)(16)(CTG)(4)), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 degrees C . Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops . Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions . Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG approximately GAC > CTG . Loops of GTC compared with CTG melt 9 degrees C lower and slip 6-fold faster . We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation.

J Biol Chem, 2000 Jun 16, 275(24), 18302 - 10
Do structural deviations between toxins adopting the same fold reflect functional differences?
Ricciardi A, le Du MH, Khayati M, Dajas F, Boulain JC, Menez A, Ducancel F.
Three-finger proteins form a structurally related family of compounds that exhibit a great variety of biological properties . To address the question of the prediction of functional areas on their surfaces, we tentatively conferred the acetylcholinesterase inhibitory activity of fasciculins on a short-chain curaremimetic toxin . For this purpose, we assimilated the three-dimensional structure of fasciculin 2 with the one of toxin alpha . This comparison revealed that the tips of the first and second loops, together with the C terminus residue, deviated most . A first recombinant fasciculin/toxin alpha chimera was designed by transferring loop 1 in its entirety together with the tip of loop 2 of fasciculin 2 into the toxin alpha scaffold . A second chimera (rChII) was obtained by adding the point Asn-61 --> Tyr substitution . Comparison of functional and structural properties of both chimeras show that rChII can accommodate the imposed modifications and displays nearly all the acetylcholinesterase-blocking activities of fasciculins . The three-dimensional structure of rChII demonstrates that rChII adopts a typical three-fingered fold with structural features of both parent toxins . Taken together, these results emphasize the great structural flexibility and functional adaptability of that fold and confirm that structural deviations between fasciculins and short-chain neurotoxins do indeed reflect functional diversity.

J Biol Chem, 2000 Jun 16, 275(24), 18180 - 7
Interaction of TAFII105 with selected p65/RelA dimers is associated with activation of subset of NF-kappa B genes; Yamit-Hezi A et al.; TAF(II)105, a substoichiometric coactivator subunit of TFIID, is important for activation of anti-apoptotic genes by NF-kappaB in response to the cytokine tumor necrosis factor (TNF)-alpha . In the present study we have analyzed the mechanism of TAF(II)105 function with respect to its regulation of p65/RelA, a component of NF-kappaB . We found two independent p65/RelA-binding domains within the N terminus of TAF(II)105 . One of these domains appears to be crucial for TAF(II)105-mediated anti-apoptotic gene activation in response to TNF-alpha . Analysis of the interaction between TAF(II)105 and different NF-kappaB complexes has revealed substantial differences in the affinity of TAF(II)105 toward different p65/RelA-containing dimers . We have identified the TNF-alpha induced anti-apoptotic A20 gene as a target gene of TAF(II)105 . A20 has a differential protective effect on cell death induced by TNF-alpha in the presence of either the dominant negative mutant of TAF(II)105 (TAF(II)105DeltaC) or the superdominant IkappaBalpha . The results suggest that the inhibitory effect of TAF(II)105DeltaC on NF-kappaB-dependent genes is restricted to a subset of anti-apoptotic genes while the effect of IkappaBalpha is more general . Thus, an interaction between NF-kappaB and a specific coactivator is important for specifying target gene activation.

J Biol Chem, 2000 Jun 16, 275(24), 18121 - 8
Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations; Zhong L et al.; Mammalian thioredoxin reductases (TrxR) are dimers homologous to glutathione reductase with a selenocysteine (SeCys) residue in the conserved C-terminal sequence -Gly-Cys-SeCys-Gly . We removed the selenocysteine insertion sequence in the rat gene, and we changed the SeCys(498) encoded by TGA to Cys or Ser by mutagenesis . The truncated protein having the C-terminal SeCys-Gly dipeptide deleted, expected in selenium deficiency, was also engineered . All three mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity with 1 mol of FAD per monomeric subunit . Anaerobic titrations with NADPH rapidly generated the A(540 nm) absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR . However, only the SeCys(498) --> Cys enzyme showed catalytic activity in reduction of thioredoxin, with a 100-fold lower k(cat) and a 10-fold lower K(m) compared with the wild type rat enzyme . The pH optimum of the SeCys(498) --> Cys mutant enzyme was 9 as opposed to 7 for the wild type TrxR, strongly suggesting involvement of the low pK(a) SeCys selenol in the enzyme mechanism . Whereas H(2)O(2) was a substrate for the wild type enzyme, all mutant enzymes lacked hydroperoxidase activity . Thus selenium is required for the catalytic activities of TrxR explaining the essential role of this trace element in cell growth.

J Biol Chem, 2000 Aug 25, 275(34), 26144 - 9
Interaction between the transcription factor SPBP and the positive cofactor RNF4 . An interplay between protein binding zinc fingers; Lyngso C et al.; The activator of stromelysin 1 gene transcription, SPBP, interacts with the RING finger protein RNF4 . Both proteins are ubiquitously expressed and localized in the nucleus . RNF4 facilitates accumulation of specific SPBP-DNA complexes in vitro and acts as a positive cofactor in SPBP-mediated transactivation . SPBP harbors an internal zinc finger of the PHD/LAP type . This domain can form intra-chain protein-protein contacts in SPBP resulting in negative modulation of SPBP-RNF4 interaction.

J Biol Chem, 2000 Aug 25, 275(34), 26523 - 9
Gene-specific silencing by expression of parallel complementary RNA in Escherichia coli; Tchurikov NA et al.; Gene-specific silencing refers to a phenomenon in which expression of an individual gene can be specifically repressed by different mechanisms on the levels of transcription, RNA splicing, transport, degradation in nuclei or cytoplasm, or blocking of translation . In different species gene-specific silencing was observed by expression or injections of antiparallel double-stranded RNA formed by a fragment of mRNA and antisense RNA . Here we show a potent and specific gene silencing in bacteria by expression of RNA, that is complementary in a parallel orientation to Escherichia coli lon mRNA . Moreover, the expression of parallel RNA is more effective at producing interference than expression of antisense RNA corresponding to the same mRNA region . Both effects of interference mediated either by parallel RNA or antiparallel RNA gradually decrease up to the 40th generation . Together with in vitro nuclease protection studies these results indicate that a parallel RNA duplex might be formed in vivo and both types of duplexes, antiparallel or parallel, can induce gene-specific silencing by similar mechanisms.

Circ Res, 2000 Jun 9, 86(11), 1146 - 52
Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca(2+) regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein; Redwood C et al.; Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in at least 8 contractile protein genes, most commonly beta myosin heavy chain, myosin binding protein C, and cardiac troponin T . Affected individuals are heterozygous for a particular mutation, and most evidence suggests that the mutant protein acts in a dominant-negative fashion . To investigate the functional properties of a truncated troponin T shown to cause HCM, both wild-type and mutant human cardiac troponin T were overexpressed in Escherichia coli, purified, and combined with human cardiac troponins I and C to reconstitute human cardiac troponin . Significant differences were found between the regulatory properties of wild-type and mutant troponin in vitro, as follows . (1) In actin-tropomyosin-activated myosin ATPase assays at pCa 9, wild-type troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition . (2) Similarly, in the in vitro motility assay, mutant troponin failed to decrease both the proportion of actin-tropomyosin filaments motile and the velocity of motile filaments at pCa 9 . (3) At pCa 5, the addition of mutant complex caused a greater increase (21.7%) in velocity of actin-tropomyosin filaments than wild-type troponin (12.3%) . These data suggest that the truncated troponin T prevents switching off of the thin filament at low Ca(2+) . However, the study of thin filaments containing varying ratios of wild-type and mutant troponin T at low Ca(2+) indicated an opposite effect of mutant troponin, causing enhancement of the inhibitory effect of wild-type complex, when it is present in a low ratio (10% to 50%) . These multiple effects need to be taken into account to explain the physiological consequences of this mutation in HCM . Further, these findings underscore the importance of studying mixed mutant:wild-type preparations to faithfully model this autosomal-dominant disease.

Plant J, 2000 Jun, 22(5), 415 - 26
Expression of Brassica juncea 3-hydroxy-3-methylglutaryl CoA synthase is developmentally regulated and stress-responsive; Alex D et al.; 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is an enzyme in mevalonate biosynthesis . In plants, investigations have focused on HMG CoA reductase (HMGR) and less is known of the preceding enzyme, HMGS . To understand the regulation of HMGS, we have isolated a Brassica juncea cDNA encoding HMGS, BjHMGS1, for use as a hybridization probe in Northern blot analyses . BjHMGS is expressed in all plant organs and shows developmental regulation in flower, seed and seedling, with highest expression in early development . In seedlings, expression is highest in young hypocotyls and is induced during the greening of etiolated cotyledons . BjHMGS is down-regulated by abscisic acid, osmotic stress and dehydration, the effects of which arrested seedling growth . Thus BjHMGS expression shows correlation with rapid cell division and growth, like HMGR . This is not unexpected, as mevalonate is the precursor to many essential isoprenoid compounds, including sterols for membrane biogenesis . Wounding, methyl jasmonate or salicylic acid induce BjHMGS expression, suggesting that, like HMGR, HMGS is involved in defence . As in animals, coordinated regulation of HMGS with HMGR occurred in B . juncea upon germination and in response to salicylic acid . HMGS assays confirmed that Escherichia coli-expressed recombinant BjHMGS1 shows HMGS activity that is inhibited by F244, a specific inhibitor of HMGS . Southern blot analysis revealed gene families encoding HMGS in Brassica species and a summation of homologous genes in the fusion amphidiploid genome of B . juncea, a bi-parental species derived from diploids B . nigra and B . campestris.

Plant J, 2000 May, 22(4), 303 - 13
Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease; Chen M et al.; Variegated plants have green- and white-sectored leaves . Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non-pigmented plastids that lack organized lamellar structures . Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned . We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities) . Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane-associated events . Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis . We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity . These include two putative ATP-binding sites . VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype . One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors . We conclude that redundant activities may be a general mechanism to explain nuclear gene-induced plant variegations.

Lett Appl Microbiol, 2000 Jun, 30(6), 427 - 31
Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions; Farnleitner AH et al.; A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions . Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients . In contrast to TTGE, DGGE displayed clear and focused bands . The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons . Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E . coli uidA sequence types.

Eur J Biochem, 2000 Jun, 267(12), 3792 - 800
The assembly pathway of outer membrane protein PhoE of Escherichia coli; Jansen C et al.; The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo . In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope . The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes . The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers . In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO) . A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates . It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein . Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane . Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands . The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO . However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton . Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.

Eur J Biochem, 2000 Jun, 267(12), 3762 - 9
Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1; Hagemeier CH et al.; Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+ . The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already . Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium . Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism . The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1) . The Km value for NAD+ was 200 microM and for NADP+ 20 microM . In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate . Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea . Despite its location, MtdB did not show sequence similarity to archaeal enzymes . The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island . Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde . On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed . We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.

Eur J Biochem, 2000 Jun, 267(12), 3661 - 71
A novel NADPH:diamide oxidoreductase activity in arabidopsis thaliana P1 zeta-crystallin; Mano J et al.; The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N, N-dimethylformamide) (diamide) to yeast {Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M . & Inze, D . (1995) J . Biol . Chem . 270, 26224}, was expressed in Escherichia coli to characterize biochemical properties of the P1-zeta-crystallin (P1-ZCr) . Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr . P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones . Two other azodicarbonyl compounds also served as substrates of P1-ZCr . Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction . Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase . The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl-reducing reaction was not . Specificity to NADPH, as judged by kcat/Km, was > 1000-fold higher than that to NADH both for quinones and diamide . N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities . Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH . The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide . Other possible physiological functions of P1-ZCr in plants are discussed.

Eur J Biochem, 2000 Jun, 267(12), 3604 - 12
Human herpes virus 8 interleukin-6 homologue triggers gp130 on neuronal and hematopoietic cells; Hoischen SH et al.; Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue . Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases . vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours . We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein . After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy . A rabbit antiserum was raised against the recombinant vIL-6 protein . vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly . Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein . vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells . Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone . These properties are important for the evaluation of the pathophysiological potential of vIL-6.

Clin Exp Allergy, 2000 Jul, 30(7), 962 - 71
Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2; Scheurer S et al.; BACKGROUND: Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients . A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen . To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen . OBJECTIVES: Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin . METHODS: Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA . Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a . The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose . Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments . RESULTS: The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa . The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens . Celery profilin was isolated as highly pure nonfusion protein . The IgE-reactivity of celery profilin was similar to that of natural protein . Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting . Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen . Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay . CONCLUSIONS: Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2 . According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4 . In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins . Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.

Br J Haematol, 2000 Apr, 109(1), 195 - 200
Effect of factor VIII concentrate on antigen-presenting cell (APC)/T-cell interactions in vitro: relevance to inhibitor formation and tolerance induction; Hodge G et al.; Inhibitor formation in patients with haemophilia receiving factor VIII (FVIII) concentrate is a common problem requiring tolerance induction therapy . Immune tolerance is dependent on defective T cell/antigen-presenting cell (APC) interactions and inhibitor antibody formation is associated with effective T-cell/B-cell interaction . We studied the expression of the cell-surface molecules involved with these interactions using multiparameter flow cytometry and a whole blood stimulation assay-phytohaemaglutinin (PHA) to activate T cells and Escherichia coli lipopolysaccharide (LPS) to activate monocytes and B cells . Up-regulation of T-cell co-stimulatory receptors CD11a, CD40 ligand (CD40L) and CTLA4 were inhibited in a dose-dependent manner by plasma-derived (pd)FVIII, but CD28 was unchanged . Up-regulation of monocyte and B-cell co-stimulatory ligands CD4O, B7-1 (CD80) and B7-2 (CD86) were also inhibited in a dose-dependent manner by pdFVIII, but LFA-3 (CD58) was unchanged . The combined inhibitory effect of prednisolone, an immunosuppressive agent used in several tolerance induction protocols, with pdFVIII on co-stimulatory molecules, was additive . There was no significant alteration in T-cell/APC adhesion or co-stimulatory molecules noted in the presence of recombinant (rh)FVIII concentrate . The inhibitory effect of pdFVIII on molecules involved in interaction between T cells and APCs may result in immune tolerance in recipients of pdFVIII concentrate . The inhibitory effect of pdFVIII on CD40/CD40L up-regulation may result in defective antibody formation . We now provide evidence that the use of pdFVIII, through interfering with APC/T-cell interactions, may be more appropriate than rhFVIII for tolerance induction.

Shock, 2000 Jun, 13(6), 459 - 63
Effects of MCI-154, a calcium sensitizer, on cardiac dysfunction in endotoxic shock in rabbits; Ming MJ et al.; This study was designed to observe the effects of MCI-154, a calcium sensitizer, on cardiac dysfunction after endotoxic shock in rabbits . Ten hours after the rabbits were given injection of 1.0 mg/kg endotoxin (Escherichia coli, O111:B4) via marginal ear veins, 0.1 mg/kg MCI-154 was injected intravenously and then 50 mL/kg normal saline (NS) + 0.1 mg/kg MCI-154 was infused continuously at a rate of 0.7 mL/min . During this process, the parameters of cardiac function were measured . It was found that 10 h after the endotoxin injection, heart rate (HR) was increased significantly while the mean arterial blood pressure (MAP), left ventricular systolic pressure (LVSP), isovolumetric pressure (IP), myocardial contractility (MC), and the area of p-dp/dt(max) vector loop (Lo) all were markedly decreased . Treatment with 50 mL/kg NS alone had slight effects on these parameters . On the contrary, LVSP, IP, MC, and Lo all were increased significantly while HR was not obviously changed and left ventricular end-diastolic pressure (LVEDP) was reduced remarkably following MCI-154 administration in endotoxic shock rabbits . The parameters of myocardial contractility were improved nearly to the values in sham shock group and were markedly higher than that in NS alone-treated group . It can be concluded that MCI-154 can exert significant therapeutic effects on cardiac dysfunction after endotoxic shock, for it improves cardiac function, dilates peripheral blood vessels, and slightly affects HR.

Mol Endocrinol, 2000 Jun, 14(6), 889 - 99
Characterization of transactivational property and coactivator mediation of rat mineralocorticoid receptor activation function-1 (AF-1); Fuse H et al.; The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor (MR) E/F domain is known to play a major role in the ligand-induced transactivation function of MR; however, it remained unclear about the transactivation function of its A/B domain . We therefore tried to characterize the MR A/B domain as the AF-1 and further studied the actions of known coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR A/B domain . Deletion analyses of rat and human MRs revealed that the A/B domains harbor a transactivation function acting as AF-1 . The MR mutant (E959Q) with a point mutation in helix 12, which causes a complete loss of MR AF-2 activity, still retained ligand-induced transactivation function, indicating a significant role for AF-1 in the full activity of the ligand-induced MR function . Among the coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two different core regions {amino acids (a.a.) 1-169, a.a . 451-603} and exhibited functional interactions with the MR A/B domain in the cultured cells . However, such interactions were undetectable in a yeast and in an in vitro glutathione-S-transferase pull-down assay, indicating that the functional interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1 activity require some unknown nuclear factor(s) or a proper modification of the A/B domain in the cells.

Microbiology, 2000 Jun, 146 ( Pt 6), 1457 - 68
The dnrO gene encodes a DNA-binding protein that regulates daunorubicin production in Streptomyces peucetius by controlling expression of the dnrN pseudo response regulator gene; Otten SL et al.; The dnrO gene is located adjacent to and divergently transcribed from the response regulator gene, dnrN, that activates the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius . Gene disruption and replacement of dnrO produced the dnrO::aphII mutant strain and resulted in the complete loss of daunorubicin biosynthesis . Suppression of the dnrO::aphII mutation by the introduction of dnrN or dnrI on a plasmid suggested that DnrO is required for the transcription of dnrN, whose product is known to be required for dnrI expression . These conclusions were supported by the effects of the dnrO mutation on expression of dnrO, dnrN and dnrI, as viewed by melC fusions to each of these regulatory genes . DnrO was overexpressed in Escherichia coli and the cell-free extract was used to conduct mobility shift DNA-binding assays . The results showed that DnrO binds specifically to the overlapping dnrN/dnrO(p1) promoter region . Thus, DnrO may regulate the expression of both the dnrN and dnrO genes.

Microbiology, 2000 Jun, 146 ( Pt 6), 1437 - 45
The Fusobacterium nucleatum porin FomA possesses the general topology of the non-specific porins; Puntervoll P et al.; FomA is a major non-specific porin of Fusobacterium nucleatum with no sequence similarity to other known porins . According to the topology model, the protein consists of 16 transmembrane beta-strands, connected by eight surface-exposed loops and seven periplasmic turns . In this study, the insertion mutagenesis approach was applied to probe the topology model . A Semliki Forest Virus (SFV) epitope was successfully inserted at 11 different sites of the FomA protein and a 6-aa insertion was successfully inserted at two different sites . Correct folding of the mutant proteins and proper incorporation into the outer membrane were assessed by heat modifiability and by an in vivo porin activity assay . Immunofluorescence microscopy analysis of intact cells, using mAbs directed against the inserted SFV epitope, revealed that three of the eight putative extracellular loops are indeed surface-exposed . Trypsin accessibility experiments confirmed the cell surface exposure of two additional loops . The results support the proposed topology model, showing that FomA possesses the general beta-barrel topology of the non-specific porins, with the interesting exception that the third loop does not seem to fulfil the role of a constriction loop.

Microbiology, 2000 Jun, 146 ( Pt 6), 1407 - 18
Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element; Souza EM et al.; The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a -12/-24 sigma(N)-dependent promoter . This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion . A 5' end to the RNA was identified at position 641, 12 bp downstream from the -12/-24 promoter . Footprinting experiments showed that the G residues at positions -26 and -9 are hypermethylated, and that the region from -10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter . In H . seropedicae nifA expression from the sigma(N)-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein . NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

Microbiology, 2000 Jun, 146 ( Pt 6), 1333 - 44
Intimin from enteropathogenic Escherichia coli mediates remodelling of the eukaryotic cell surface; Phillips AD et al.; Adhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton . Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion . A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation . In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence . Intimin appears to play a key role in MLP elongation . At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected . In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of 'cage-like' structures engulfing the bacteria . Inoculating HEp-2 cells with intimin-coated latex spheres induced similar 'cage-like' structures . Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred . Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e . elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre . These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.

Microbiology, 2000 Jun, 146 ( Pt 6), 1275 - 86
Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria; Iteman I et al.; PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium NOSTOC: PCC 7120 gave three products . Two represented true ITS regions of different sizes, while the third was a heteroduplex . The longer spacer (ITS-L) contained 512 nucleotides and carried tRNA(Ile) and tRNA(Ala) genes, separated by a large stem-loop structure (V2) composed of short tandemly repeated repetitive sequences . Both tRNA genes, and the 5' half of the intervening stem, were absent from the shorter spacer (ITS-S), of length 283 nucleotides, which was otherwise almost completely identical to ITS-L . The two spacer regions of NOSTOC: PCC 7120 were aligned to published ITS sequences of cyanobacteria, the cyanelle of Cyanophora paradoxa and Escherichia coli . Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNA(Ile) and tRNA(Ala) genes, only the tRNA(Ile) gene, or neither, there is no correlation between ITS size and coding capacity for tRNAs . Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E . coli . Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified, and regions homologous to bacterial antiterminators (box B-box A) were located . The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.

J Biol Chem, 2000 Sep 15, 275(37), 29061 - 5
Role of activating region 1 of Escherichia coli FNR protein in transcription activation at class II promoters; Wing HJ et al.; FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase . At class II FNR-dependent promoters, where the DNA site for FNR overlaps the -35 element, activating region 1 of FNR is proposed to interact with the C-terminal domain of the RNA polymerase alpha-subunit . Using a model class II FNR-dependent promoter, FF(-41.5), we have performed in vivo and in vitro experiments to investigate the role of this interaction . Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription . Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation . A new method for the purification of the FNR protein is also described.

Indian J Biochem Biophys, 1999 Dec, 36(6), 429 - 32
Cloning, expression and purification of the DNA binding domain of RFX protein; Panchal SC et al.; The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimer DNA binding proteins which have diverse regulatory functions in eukaryotic organisms . To characterize this novel motif, a 78mer polypeptide corresponding to the DBD of human hRFX (hrfX1/DBD), a prototypical member of the RFX family has been cloned and overproduced in Escherichia coli . A purification procedure using cation exchange chromatography has also been developed.

Mol Microbiol, 2000 Jun, 36(5), 1101 - 12
Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway; Dassler T et al.; A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain . Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible . Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein . Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O-acetyl-L-serine, especially in the presence of thiosulphate . Most of the cysteine was found to be masked in 2-methyl-2,4-thiazolidinedicarboxylic acid . N-acetyl-L-serine was also present in the medium, but it is open to question whether it represents a primary excretion product . Measurement of the induction status of the cysteine regulon by means of a cysK'-'lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency . The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway . Its relation to other efflux systems and the physiological role are discussed.

Mol Microbiol, 2000 May, 36(4), 982 - 94
Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli; Mohanty BK et al.; Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single-stranded substrates that facilitate the degradation of mRNAs by ribonucleases . Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E . The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels . RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts . In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3' termini that serve as substrates for PAP I . In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced . Conversely, toxicity is significantly reduced in the presence of excess RNase II . Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.

Mol Microbiol, 2000 May, 36(4), 973 - 81
Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli; Hendricks EC et al.; We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes . These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology . Growth was studied in synchronous cultures and in microcolonies derived from single cells . SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes . This has been called guillotining . Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side . Growth of these filaments was severely reduced in hipA+ strains . We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth . The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA-negative cells . If SOS induction was blocked by lexA3 (Ind-), filaments did not form, and cell division was not inhibited . However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.

Mol Microbiol, 2000 May, 36(4), 926 - 39
PEST sequences in cAMP-dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases; Borges AC et al.; During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels . The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B . emersonii R and C subunits . Deletions in the PEST sequences were obtained by site-directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli . Proteolysis assays of the various R and C recombinant forms, using B . emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation . Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions . The PEST sequence in B . emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein . C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability . These results suggest that the N-terminal extension may only play a role in C subunit degradation.

Mol Microbiol, 2000 May, 36(4), 913 - 25
Suppression of a DnaX temperature-sensitive polymerization defect by mutation in the initiation gene, dnaA, requires functional oriC; Blinkova A et al.; Temperature sensitivity of DNA polymerization and growth, resulting from mutation of the tau and gamma subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA . These mutations simultaneously cause defective initiation at 20 degrees C . Efficient suppression, defined as restoration of normal growth rate at 39 degrees C to essentially all the cells, depends on functional oriC . Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression . This suggests that the suppression mechanism depends on reduced activity of DnaACs, Sx . Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.

Mol Microbiol, 2000 May, 36(4), 898 - 912
The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage beta-1,3/beta-1, 4-glucan; Meissner K et al.; Thermotoga maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1-C2) . Its catalytic domain (-B-) is flanked by duplicated non-catalytic domains . The C-terminal repeated domains represent cellulose-binding domains (CBDs) . Xylanase domains related to the N-terminal domains of XynA (A1-A2) are called thermostabilizing domains because their deletion normally leads to increased thermosensitivity of the enzymes . It was found that a glutathione-S-transferase (GST) hybrid protein (GST-A1A2) containing both A-domains of XynA can interact with various soluble xylan preparations and with mixed-linkage beta-1,3/beta-1,4-glucans . GST-A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST-C2, which contains the C-terminal CBD of XynA, did not interact with soluble xylan . Another hybrid protein, GST-A2, displayed the same binding properties as GST-A1A2, indicating that A2 alone can also promote xylan binding . The dissociation constants for the binding of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose by GST-A2, as determined at 20 degrees C by fluorescence quench experiments, were 8.1 x 10(-3) M, 2.3 x 10(-4) M, 2.3 x 10(-5) M, 2.5 x 10(-6)M and 1.1 x 10(-6) M respectively . The A-domains of XynA, which are designated as xylan binding domains (XBD), are, from the structural as well as the functional point of view, prototypes of a novel class of binding domains . More than 50 related protein segments with hitherto unknown function were detected in about 30 other multidomain beta-glycanases, among them putative plant (Arabidopsis thaliana) xylanases . It is argued that polysaccharide binding and not thermostabilization is the main function of A-like domains.

Mol Microbiol, 2000 May, 36(4), 806 - 16
PAS domain residues involved in signal transduction by the Aer redox sensor of Escherichia coli; Repik A et al.; PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation . Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL . We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli . Forty-two residues in Aer were substituted using cysteine-replacement mutagenesis . Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen . Four of them also led to the loss of the non-covalently bound FAD cofactor . Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal-on bias . One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa . Residues critical for signalling were mapped onto a three-dimensional model of the Aer PAS domain, and an FAD-binding site and 'active site' for signal transduction are proposed.

Mol Microbiol, 2000 May, 36(3), 651 - 61
Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate; Murray M et al.; Inositol monophosphatase plays a vital role in the de novo biosynthesis of inositol and in the phosphoinositide second messenger signalling pathway . We cloned the Saccharomyces cerevisiae open reading frame (ORF) YHR046c (termed INM1), which encodes inositol monophosphatase, characterized the protein Inm1p and analysed expression of the INM1 gene . INM1 was expressed in bacteria under the control of the lacZ promoter . The purified protein has inositol monophosphatase activity that is inhibited by the antibipolar drug lithium, but not valproate . In the inm1Delta:URA3 null mutant, inositol monophosphatase activity was reduced but not eliminated . The disruption had little effect on growth in the presence of lithium or valproate and no effect on growth in the absence of inositol . To characterize the regulation of INM1, we examined the effects of inositol, carbon source, growth phase, and the antibipolar drugs lithium and valproate on INM1 expression using an INM1-lacZ reporter gene . Unlike all other phospholipid biosynthetic enzyme-encoding genes studied, which contain the UASINO regulatory element, INM1 expression is increased in the presence of inositol . In addition, INM1 expression was repressed during growth in glycerol and derepressed as glucose-grown cells entered stationary . Both lithium and valproate, which cause a decrease in intracellular inositol, effect a decrease in INM1 expression . A model is presented to account for regulation of INM1 expression.

Mol Microbiol, 2000 May, 36(3), 557 - 69
Analysis of the DNA-binding domain of Escherichia coli DnaA protein; Blaesing F et al.; The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467) . The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain . In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection . Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C . Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins . No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440 . Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C . The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures . dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.

Mol Microbiol, 2000 May, 36(3), 528 - 38
Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for holliday junction branch migration; Iwasaki H et al.; Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair . The RuvB molecular motor is an intrinsic ATP-dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class . In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins . Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.

Bioorg Med Chem Lett, 2000 May 15, 10(10), 1125 - 7
Syntheses and properties of photoactivatable sugar derivatives designed to probe the sugar-binding site of melibiose permease; Ambroise Y et al.; Three new photoreactive sugar analogues bearing an azido, a diazonium salt or a diazirine group as the photophore as well as a tritium atom were developed . Two of these new photoactivatable compounds gave excellent preliminary results, with a high affinity for the melibiose permease of Escherichia coli.

Proteins, 2000 Aug 1, 40(2), 185 - 92
Empirical calculation of the relative free energies of peptide binding to the molecular chaperone DnaK; Kasper P et al.; We describe a methodology to calculate the relative free energies of protein-peptide complex formation . The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions . A free energy-surface area relationship served to calculate the nonpolar free energy term . The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains . We applied this methodology to a series of DnaK*peptide complexes . On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes . Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy . The calculated relative binding free energies were compared with the experimentally determined free energies . Linear scaling of the calculated terms was applied to fit them to the experimental values . The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86 . The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues . Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand . Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation . The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available .

Muscle Nerve, 2000 Jun, 23(6), 967 - 9
Induction of the ATP-sensitive potassium (uK(ATP)-1) channel by endotoxemia; Czaika G et al.; The effect of sepsis on the ubiquitously expressed ATP-sensitive potassium (uK(ATP)-1) channel expression was measured in Sprague-Dawley rat diaphragms . Rats were treated with either 0.5 ml saline or 20 mg/Kg E . coli lipopolysaccharides and sacrificed at 3, 6, 12, 24, or 48 h later . Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that channel mRNA expression was increased at 3 h and continued to rise up to 48 h . Western blotting analysis showed a approximately 9-fold increase in channel protein expression 24 h after sepsis . Our results demonstrate that sepsis upregulates the uK(ATP)-1 channel .

J Biol Chem, 2000 Sep 1, 275(35), 26834 - 41
Doppel is an N-glycosylated, glycosylphosphatidylinositol-anchored protein . Expression in testis and ectopic production in the brains of Prnp(0/0) mice predisposed to Purkinje cell loss; Silverman GL et al.; The Prnd gene encodes a homolog of the cellular prion protein (PrP(C)) called doppel (Dpl) . Up-regulation of Prnd mRNA in two distinct lines of PrP gene ablated (Prnp(0/0)) mice, designated Rcm0 and Ngsk, is associated with death of Purkinje cells . Using recombinant Dpl expressed in Escherichia coli and mouse neuroblastoma cells we demonstrate that wild type (wt) Dpl, like PrP(C), adopts a predominantly alpha-helical conformation, forms intramolecular disulfide bonds, has two N-linked oligosaccharides, and is presented on the cell surface via a glycosylphosphatidylinositol anchor . Dpl protein was detected in testis of wt mice . Using Triton X-114 phase partitioning to enrich for glycosylphosphatidylinositol-anchored proteins, Dpl was detected in brain samples from Rcm0 Prnp(0/0) mice but was absent in equivalent samples from wt mice and ZrchI Prnp(0/0) mice, indicating that ectopic expression of this protein may cause cerebellar pathology in Rcm0 mice . Biochemical and structural similarities between PrP(C) and Dpl documented here parallel the observation that ataxic Ngsk Prnp(0/0) mice can be rescued by overexpression of wild-type PrP transgenes, and suggest that cell surface PrP(C) can antagonize the toxic effect of Dpl expressed in the central nervous system.

FEMS Microbiol Rev, 2000 Jul, 24(3), 303 - 16
Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli; Fabianek RA et al.; Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds . In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway . Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site . Most of them have a domain that displays the thioredoxin-like fold . In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity . Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors . This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E . coli.

Biochemistry, 2000 Jun 13, 39(23), 6969 - 78
Catalytic cysteine of thymidylate synthase is activated upon substrate binding; Phan J et al.; The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies . Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis . Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E . coli in medium lacking thymidine . Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme . Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher . The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively . Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme . Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme . S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C) . The high-resolution data sets were refined to a R(crys) of 19.9% . In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine . The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines . It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.

Biochemistry, 2000 Jun 13, 39(23), 6891 - 7
Independent functions for the N- and C-termini in the overlap region of tropomyosin; Moraczewska J et al.; Tropomyosin (TM) is a coiled-coil that binds head-to-tail along the helical actin filament . The ends of 284-residue tropomyosins are believed to overlap by about nine amino acids . The present study investigates the function of the N- and C-terminal overlap regions . Recombinant tropomyosins were produced in Escherichia coli in which nine amino acids were truncated from the N-terminal, C-terminal, or both ends of striated muscle alpha-tropomyosin (TM9a) and TM2 (TM9d), a nonmuscle alpha-tropomyosin expressed in many cells . The two isoforms are identical except for the C-terminal 27 amino acids encoded by exon 9a (striated) or exon 9d (TM2) . Removal of either end greatly reduces the actin affinity of both tropomyosins in all conditions and the cooperativity with which myosin promotes tropomyosin binding to actin in the open state . N-Terminal truncations generally are more deleterious than C-terminal truncations . With TM9d, truncation of the N-terminus is as deleterious as both for myosin S1-induced binding . None of the TM9d variants binds well to actin with troponin (+/-Ca(2+)) . TM9a with the truncated N-terminus binds more weakly to actin with troponin (-Ca(2+)) than when the C-terminus is removed but more strongly than when both ends are removed; the actin binding of all three forms is cooperative . The results show that the ends of TM9a, though important, are not required for cooperative function and suggest they have independent functions beyond formation of an overlap complex . The nonadditivity of the TM9d truncations suggests that the ends may primarily function as a complex in this isoform . A surprising result is that all variants bound with the same affinity, and noncooperatively, to actin saturated with myosin S1 . Evidently, end-to-end interactions are not required for high-affinity binding to acto-myosin S1.

Biochemistry, 2000 Jun 13, 39(23), 6857 - 63
Stoichiometry of complex formation between Copper(I) and the N-terminal domain of the Menkes protein; Cobine PA et al.; The inherent cellular toxicity of copper ions demands that their concentration be carefully controlled . The cellular location of the Menkes ATPase, a key element in the control of intracellular copper, is regulated by the intracellular copper concentration through the N-terminus of the enzyme, comprising 6 homologous subdomains or modules, each approximately 70 residues in length and containing a -Cys-X-X-Cys- motif . Based on the proposal that binding of copper to these modules regulates the Menkes ATPase cellular location by promoting changes in the tertiary structure of the enzyme, we have expressed the entire N-terminal domain (MNKr) and the second metal-binding module (MNKr2) of the Menkes protein in E . coli and purified them to homogeneity . Ultraviolet-visible, luminescence, and X-ray absorption spectroscopy show that copper and silver bind to the single module, MNKr2, with a stoichiometry of one metal ion per module . However, the array of six modules, MNKr, binds Cu(I) to produce a homogeneous conformer with 4 mol equiv of metal ion . The metal ions are bound in an environment that is shielded from solvent molecules . We suggest a model of the Menkes protein in which the Cu(I) binding induces tertiary changes in the organization of the six metal-binding domains.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6451 - 6
Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana; Rohdich F et al.; A hypothetical gene with similarity to the ispD gene of Escherichia coli was cloned from Arabidopsis thaliana cDNA . The ORF of 909 bp specifies a protein of 302 amino acid residues . The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78-202 bp . A fragment comprising amino acid residues 76-302 was expressed in a recombinant E . coli strain . The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl-d-erythritol from 2C-methyl-d-erythritol 4-phosphate with a specific activity of 67 micromol small middle dotmin(-1) mg(-1) . The Michaelis constants for 4-diphosphocytidyl-2C-methyl-d-erythritol and CTP were 500 microM and 114 microM, respectively.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6298 - 305
Inaugural article: retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins; Lombardi A et al.; De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins . The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites . The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 A rms deviation by using a simple geometric model with only six adjustable parameters . These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins . This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 A . This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs . The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands . The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands . In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins . DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7399 - 404
The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair; Amundsen SK et al.; The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli . The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGG-3') . It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity {Thaler, D . S., Sampson, E., Siddiqi, I., Rosenberg, S . M., Stahl, F . W . & Stahl, M . (1988) in Mechanisms and Consequences of DNA Damage Processing, eds . Friedberg, E . & Hanawalt, P . (Liss, New York), pp . 413-422; Churchill, J . J., Anderson, D . G . & Kowalczykowski, S . C . (1999) Genes Dev . 13, 901-911} . We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recB(D1080A)CD . We report here that the resulting strain, recB(D1080A)C, was proficient for recombination and DNA repair . Recombination proficiency was accompanied by a change in enzyme activity: RecB(D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB(D1080A)CD enzyme did not . Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E . coli cells and suggest that RecD interferes with the enzyme domain required for its loading . A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading . Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7112 - 7
Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein; Brill LM et al.; The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta . To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography . Soluble MP was maintained at >4 mg/ml without aggregation and displayed approximately 70% alpha-helical conformation in the presence of urea and SDS . A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin . Two hydrophobic regions within the core were highly resistant to proteolysis . Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative alpha-helical transmembrane domains and a protease-sensitive carboxyl terminus.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7248 - 53
DNA delivery by phage as a strategy for encapsulating toroidal condensates of arbitrary size into liposomes; Lambert O et al.; We report a strategy for encapsulating and condensing DNA . When T5 phage binds to its membrane protein receptor, FhuA, its double stranded DNA (120,000 bp) is progressively released base pair after base pair in the surrounding medium . Using cryoelectron microscopy, we have visualized the structures formed after T5 phage DNA is released into neutral unilamellar proteoliposomes reconstituted with the receptor FhuA . In the presence of spermine, toroidal condensates of circumferentially wrapped DNA were formed . Most significantly, the sizes of these toroids were shown to vary, from 90 to 200 nm in their outer diameters, depending on the number of DNA stands transferred . We have also analyzed T5 DNA release in bulk solution containing the detergent-solubilized FhuA receptor . After DNA release in a spermine containing solution, huge DNA condensates with a diameter of about 300 nm were formed containing the DNAs from as many as 10-20 capsids . At alkaline pH, the condensates appeared as large hollow cylinders with a diameter of 200 nm and a height of 100-200 nm . Overall, the striking feature of our experiments is that, because of the progressive release of DNA from the phage capsid, the mechanism of toroid formation is fundamentally different from that in the classical studies in which highly dilute, "naked" DNA is condensed by direct addition of polyvalent cations; as a consequence, our method leads to toroids of arbitrary size.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7393 - 8
Transposon stability and a role for conjugational transfer in adaptive mutability; Godoy VG et al.; Lac(+) revertants of Escherichia coli that occur after prolonged nonlethal selection display a high frequency of transposon loss when the transposon Tn10 and the reverting lacI33 allele are linked on an F'128 episome . As many as 20% of the Lac(+) revertants are sensitive to tetracycline, about half because of transposon loss, nearly all by precise excision, and the remainder because of amplification of both the transposon and the linked lac allele . Lethality of the amplified products in the presence of tetracycline is a peculiarity of the tetA gene at high gene dosage . The selective conditions on lactose medium result in 10% transposon-free revertants, whether or not a requirement for conjugal DNA transfer is imposed . In addition, a similar fraction, about 5% of Lac(-) unreverted colonies that are products of transfer between cells experiencing nonlethal selection are also tetracycline-sensitive, and all are attributable to loss of the Tn10 transposon . These results suggest the possibility that the high frequency of transposon loss is a consequence of conjugal transfer, making this loss a marker for that transfer . We suggest that conjugal DNA transfer may be a prominent feature in the mutability process that occurs during nonlethal selection and that the subset of bacteria displaying hypermutability are those that experience such transfer.

J Biol Chem, 2000 Aug 18, 275(33), 25427 - 35
Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism; Bai J et al.; The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis . Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B . We have determined how deep the Ca(2+)-binding loops of Ca(2+).C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca(2+)-triggered interactions with the SNARE complex . To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes . Kinetics experiments revealed that cis interactions are rapid (< or =500 micros) . Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles . Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether . C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles . This novel mode of clustering may allow for rapid Ca(2+)-triggered oligomerization of the protein via the membrane distal C2B domain.

J Biol Chem, 2000 Aug 18, 275(33), 25342 - 50
beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf; Schmitt JM et al.; G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade . Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases . In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs) . Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR . This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required . Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms . beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf . beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling . We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.

J Protein Chem, 1999 Nov, 18(8), 823 - 30
Fluorescence quenching studies of Trp repressor-operator interaction; Blicharska Z et al.; Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding . Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components . The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern-Volmer quenching constant equal to about 2.0-2.3 M(-1) . The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern-Volmer quenching constant characteristic of L-tryptophan in the solvent . When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed . Acrylamide quenching experiments indicate that the Stern-Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M(-1) . The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.

J Protein Chem, 1999 Nov, 18(8), 813 - 21
Utilization of the Streptoalloteichus hindustanus resistance determinant ShBle as a protein framework: effect of mutation upon ShBle dimerization and interaction of C-terminal displayed peptide epitopes; Nuttall SD et al.; We have selected the Streptoalloteichus hindustanus bleomycin-resistance protein ShBle, a 28-kDa homodimer, as a scaffold for the display of bioactive peptides and other peptide epitopes . To create a monomeric scaffold, we investigated the effect of mutating residue proline 9 to glycine . This residue plays a critical role in ShBle dimerization by affecting the position of the eight N-terminal residues which secure the interaction between the monomeric subunits . We demonstrate that this mutation weakens the dimerization interaction, resulting in establishment of a stable equilibrium between monomeric and dimeric ShBle species in solution . Circular dichroism and SDS-PAGE data indicate that the Pro9Gly mutation does not disrupt the structure of the molecule . Production of a fully monomeric form of ShBle required complete removal of the eight-residue N-terminal peptide, and the interaction across the now solvent-exposed hydrophobic interface of the ShBle monomer was insufficient to drive dimerization . To demonstrate efficient display of epitope tags on the ShBle protein, we displayed dual-octapeptide FLAG tags at the protein C-terminus . These additions did not interfere with protein folding or activity . The resulting ShBle scaffold was used to compare the efficiency of two commercial FLAG-specific antibodies by biosensor.

J Immunother, 2000 May-Jun, 23(3), 379 - 86
Development of a polynucleotide vaccine from melanoma antigen recognized by T cells-1 and recombinant protein from melanoma antigen recognized by T cells-1 for melanoma vaccine clinical trials; Lee SW et al.; MART-1, a melanoma antigen recognized by T cells-1, is a melanocyte lineage-differentiation antigen expressed only in melanocytes and melanoma cells . This protein is recognized by many T-lymphocyte lines that are human leukocyte antigen (HLA)-A2 restricted and melanoma reactive . These observations have culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides . Polynucleotide immunization is a promising alternative to recombinant viral vaccines that allows delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity . In preparation for a phase I clinical trial of MART-1 polynucleotide immunization in patients with resected melanoma who were at significant risk for recurrence, the authors constructed a plasmid DNA encoding the MART-1 cDNA under transcriptional regulatory control of the cytomegalovirus immediate early promoter-enhancer and partially deleted intron A . This plasmid directs high-level MART-1 expression in transduced myoblasts and maturing myocytes diffusely throughout the cytoplasm . Immunization of mice with this construct by intramuscular injection elicited MART-1-specific immune responses in all animals . Previous trials of MART-1 immunization have been unable to examine the humoral immune response to MART-1 because of a lack of sufficient, highly purified protein . We have produced and purified Escherichia coli recombinant MART-1 protein using a glutathione-S-transferase fusion protein expression system . Protein staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of MART-1 protein at approximately 20 kD; and Western immunoblotting with an anti-MART-1 monoclonal antibody confirmed a doublet at approximately 20 kD . These findings are consistent with previous reports using different expression systems for recombinant MART-1 . This protein preparation functioned well in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MART-1 antibody responses in a mouse model; and a panel of healthy donor human sera showed minimal binding to ELISA plates coated with the protein, supporting its utility in monitoring human anti-MART-1 antibody responses . The glutathione-S-transferase fusion method yielded approximately 200 micrograms MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plates.

Mol Biochem Parasitol, 2000 May, 108(2), 207 - 16
Molecular cloning and immunological characterization of phosphoglycerate kinase from Clonorchis sinensis; Hong SJ et al.; The parasite Clonorchis sinensis was determined to utilize a large amount of external glucose to carry its energy metabolism . Phosphoglycerate kinase (PGK), a glycolytic enzyme, found in many parasites, has been identified as one of the candidate molecules distinguished from human counterparts for vaccine and drug developments . A cDNA clone purified by screening a C . sinensis cDNA library using a heterologous cDNA probe encoded a putative peptide of 415 amino acids with over 60% identities with PGKs from a number of animals . The putative peptides revealed domains corresponding to 12 beta-sheets and inner loops forming a substrate-binding cleft of animal PGKs . The gene product was overexpressed in Escherichia coli and showed a PGK-like enzyme activity . A polyclonal antibody raised against the recombinant C . sinensis PGK was specific to native C . sinensis PGK and localized it to the muscular tissue and tegument of the adult flukes . The C . sinensis PGK elicited antibodies in C . sinensis-infected rabbits . Therefore, it is proposed that C . sinensis PGK could be used as an immunoreagent in the serodiagnosis for clonorchiasis.

Acta Trop, 2000 May 31, 75(3), 331 - 40
Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation; Rott MB et al.; Two different Echinococcus granulosus antigen B subunits (AgB8/1 and AgB8/2) were characterized and the structure of the genes encoding these two proteins were compared . DNA sequences were expressed in Escherichia coli and the antigens' diagnostic value was then assessed . The genomic sequence of AgB8/1 has a 92 bp intron in the position corresponding to amino acid 16; the AgB8/2 genomic sequence presents a 68 bp intron in the position corresponding to amino acid 20 . Both introns are located between the putative N-terminal hydrophobic sequence and the secreted peptide . A comparison between the AgB8/1 and AgB8/2 nucleotide sequences showed a 53.5% identity among exons and a 50% identity between introns . According to the molecular diversity analysis, the elapsed time since both genes shared a common ancestor would be around 4.2x10(7) years . When the native AgB and the two recombinant antigens (rAgB8/1 and rAgB8/2) were tested in an anti-IgG ELISA, the sensitivity of the native antigen B was 77.41% and its specificity was 81.9%, while rAgB8/1 showed 54.84% of sensitivity and 80.17% of specificity and rAg138/2 had an 83.87% sensitivity and a 98.28% specificity . Statistical analysis confirms that rAgB8/2 has a better performance than rAgB8/1 and native AgB in ELISA.

Mutat Res, 2000 Jul, 463(1), 1 - 12
The origins of the back-mutation assay method: a personal recollection; Grigg GW; The back-mutation assay method for determining the mutagenicity of various treatments was first developed a little over 50 years ago and has been in continuous use ever since . Shortly after the method was first used it became evident that certain factors of cell density, composition of media, etc., had to be carefully controlled to preserve an acceptable reliability of the method . A factor of particular importance was the suppression of growth of back-mutant prototrophic cells by the large number of auxotrophic cells present, a phenomenon which later became known as the "Grigg Effect." This review describes the origins of the back-mutation method and of the confounding competitive suppression phenomenon, the cause of competitive suppression, methods of diagnosing whether it is likely to bias the interpretation of a particular back-mutation experiment, and an experimental design which removes it entirely as a possible source of error . A number of other phenomena, such as phenotypic lag and coincident mutation associated with back-mutation, are also discussed as possible sources of error.

Mutat Res, 2000 May 8, 467(2), 129 - 38
Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains; Salmelin C et al.; Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and its biologically active beta-oxidation product phenylacetic acid mustard (PAM; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromatic alkylators . CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant . The chemical structures indicate that CLB and PAM are mutagenic, teratogenic and carcinogenic, but the mode of action has remained obscure . We have investigated the biological effects of CLB and PAM with DNA repair-deficient Escherichia coli strains . In contrast to MNNG (N-methyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E . coli, but permeabilization of the outer membrane of the cells through use of polymyxin B nonapeptide (PMBN) rendered them susceptible to these compounds . The importance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacking O(6)-methylguanine-DNA methyltransferase I and II (ada ogt) . Similarly, the protective role of base excision repair (BER) was substantiated by demonstration of an even more increased susceptibility to CLB and PAM of cells lacking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1) . Cells deficient in mismatch repair (mutS) appeared to be slightly more sensitive than normal cells to CLB and PAM, although no such sensitivity to MNNG was observed . This implicates the role of mismatches in CLB- and PAM-related cytotoxicity . It is generally believed that bifunctional alkylating agents, like CLB and PAM, exert their cytotoxic action via DNA cross-linking . Our results with O(6)-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient cells indicate that removal of the adducts prior to the formation of cross-links is an important mechanism maintaining cell viability . We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E . coli cells, whose outer membrane is not naturally permeable to mutagens or other interesting compounds.

Biochim Biophys Acta, 2000 Jun 15, 1501(2-3), 219 - 26
Characterization of an 84 kDa protein inducing an immediate hypersensitivity reaction in rabbits sensitized to Haemaphysalis longicornis ticks; Mulenga A et al.; An immunogenic 84 kDa protein was isolated and purified from whole tick extracts of Haemaphysalis longicornis larvae by a combination of ion exchange, reverse phase and hydrophobic interaction chromatographies . The protein, when injected intradermally into rabbits exposed to repeated tick feeding, induces an immediate cutaneous hypersensitivity reaction . It has been purified to homogeneity as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining . Amino acid sequences for two peptides derived from proteolytic cleavage of p84 were scanned against known proteins on the SWISS-PROT database . A 7 residue motif, ISGWGNT present in one of the two peptides appeared conserved in both vertebrate and invertebrate trypsin-like serine proteinases, while another 7 amino acid motif, HVPAGQI present in the second peptide showed homology to an Escherichia coli ATP-binding protein . We have discussed our findings in relation to isolation and characterization of target antigens for tick vaccine candidates.

Biochim Biophys Acta, 2000 Jun 15, 1501(2-3), 116 - 24
Apparent cooperativity in multivalent verotoxin-globotriaosyl ceramide binding: kinetic and saturation binding studies with {(125)I}verotoxin; Peter MG et al.; Verotoxin (VT) binding to the trisaccharide portion of globotriaosyl ceramide (Gb(3)) is believed to be a crucial step in the development of hemolytic uremic syndrome (HUS) commonly known as 'Hamburger disease' . This interaction is the initial step in the binding process and defines the specificity of verotoxin binding to cellular membranes . Although molecular modeling, co-crystallization and co-NMR studies with VT and the trisaccharide moiety of Gb(3) have indicated potential multiple sites for Gb(3) binding, little is known about their direct effects on kinetic and equilibrium binding . Here we describe how the binding of radiolabeled VT ({(125)I}VT1) to Gb(3) in a microtiter well format, is driven by two different association rate constants (k(+1a)=0.0075 and k(+1b)=0.275 min(-1) nM(-1)) with the high affinity site representing 15% of the total specific binding sites . Binding was reversible at room temperature, reached equilibrium after 2-3 h, and non-specific binding was less than 5% . Equilibrium binding studies defined by {(125)I}VT1 saturation binding to 15, 30, 60 and 120 ng Gb(3)/well, showed the presence of a single site with dissociation constants (K(d)s) ranging between 0.5 and 3 nM . However, the maximum density of specific {(125)I}VT1 binding sites (B(max)) did not directly correlate with the Gb(3) concentration per well: the most{(125)I}VT1 binding was observed for 60 ng Gb(3) (B(max)=1.28 nM; compared to 0 . 23 nM for 30 ng Gb(3) and 0.65 nM for 120 ng Gb(3)) . Furthermore, while Hill coefficients (n(H)) for 15, 30 and 120 ng Gb(3) were close to unity indicating single interactions, for the saturation isotherm for 60 ng Gb(3)/well n(H) was 1.4 . Subsequent Scatchard analysis yielded a concave downward curve for {(125)I}VT1 binding to 60 ng Gb(3)/well, suggesting positive co-operativity . We present, for the first time, conclusive binding data confirming the presence of at least two discrete Gb(3) binding sites: these multivalent interactions between verotoxin VT-1 and Gb(3) were described by association reactions driven by two distinct rate constants, as well as by the positive co-operativity governing binding at a restricted receptor concentration . These results imply that the concentration of Gb(3) on the surface of target cells can have a complex, non-linear effect on verotoxin binding and thereby, on sensitivity to cytotoxicity.

Mutat Res, 2000 May 30, 450(1-2), 181 - 92
Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice; Murai H et al.; We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer . To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (-/-), (+/-) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently . The XPA (-/-) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m(2)) of UVB-irradiation than the XPA (+/-) and (+/+) mice, while, at a high dose (900 J/m(2)) they showed almost the same frequency of mutation as the XPA (+/-) and (+/+) mice, probably because of cell death in the epidermis of the XPA (-/-) mice . However, CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (-/-) mice than the XPA (+/-) and (+/+) mice at both doses of UVB . This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.

Mutat Res, 2000 May 30, 450(1-2), 95 - 105
Mutations induced by reactive nitrogen oxide species in the supF forward mutation assay; Routledge MN; Nitric oxide is an important bioregulatory molecule with a range of physiological functions . Nitric oxide can also react with oxygen species to produce a range of reactive nitrogen oxides that can damage DNA and lead to mutations of the DNA base sequence . The mutagenicity of a variety of reactive nitrogen oxide species and related DNA damaging agents in the supF assay are reviewed here, in the context of recent reports that relate to the nature of the DNA lesions responsible for the induced mutations . Mutations induced by nitric oxide in the supF assay are compared to those induced by N(2)O(3), nitrous acid, peroxynitrite and different reactive oxygen species . The effect of replication of the damaged pSP189 plasmid in human cells or Escherichia coli cells is also considered.

Mutat Res, 2000 May 30, 450(1-2), 61 - 73
Mutation spectra in supF: approaches to elucidating sequence context effects; Canella KA et al.; Shuttle vectors carrying the supF suppressor tRNA gene were originally developed for mutagenesis experiments in primate and human cells . Since then, the supF gene has been used as a mutation reporter in other mammalian cells, yeast, Escherichia coli, and transgenic mice . The widespread use of the vector for studies of many DNA reactive agents has produced a large database of mutation spectra . These provide primary information on the kinds and distribution of mutations provoked by many agents and, in many instances, allow comparisons between related agents or the same agent in different cell backgrounds . In this review we will discuss some of these data with a primary focus on the interpretation of UV mutation spectra . We will also describe our development and application of custom supF marker genes as an approach to studying the effect of sequence context on mutation hotspots and cold spots . Our studies suggest that C-C photoproducts are not mutagenic in certain sequence contexts in which T-C photoproducts are mutation hotspots . In addition, we have found several examples of sequence context effects acting as much as 80 bases away from the site of mutation . We will consider some of the problems raised by these studies and the possible resolution of some of them offered by the newly discovered family of damage bypass DNA polymerases.

Mutat Res, 2000 May 30, 450(1-2), 19 - 40
In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in Escherichia coli; Chandrasekhar D et al.; In vivo formation and repair of the major UV-induced DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs), have been examined at the gene and nucleotide level in Escherichia coli . Each type of DNA photoproduct has individually been studied using photoreactivation and two newly developed assays; the multiplex QPCR assay for damage detection at the gene level and the reiterative primer extension (PE) assay for damage detection at the nucleotide level . In the E . coli lacI and lacZ genes, CPDs and 6-4 PPs form in a 2:1 ratio, respectively, during UV irradiation . Repair of 6-4 PPs is more efficient than repair of CPDs since, on the average, 42% of 6-4 PPs are repaired in both genes in the first 40 min following 200 J/m(2) UV irradiation, while 1% of CPDs are repaired . The location, relative frequency of formation, and efficiency of repair of each type of photoproduct was examined in the first 52 codons of the E . coli lacI gene at the nucleotide level . Hotspots of formation were found for each type of lesion . Most photoproducts are at sites where both CPDs and 6-4 PPs are formed . Allowing 40 min of recovery following 200 J/m(2) shows that in vivo repair of 6-4 PPs is about fourfold more efficient than the repair of CPDs . Comparison of the lesion-specific photoproduct distribution of the lacI gene with a UV-induced mutation spectrum from wild-type cells shows that most mutational hotspots are correlated with sites of a majority of CPD formation . However, 6-4 PPs are also formed at some of these sites with relatively high frequency . This information, taken together with the observation that 6-4 PPs are repaired faster than CPDs, suggest that the cause of mutagenic hotspots in wild-type E . coli is inefficient repair of CPDs.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 457 - 66
A model for the structure of subunit a of the Escherichia coli ATP synthase and its role in proton translocation; Vik SB et al.; Most of what is known about the structure and function of subunit a, of the ATP synthase, has come from the construction and isolation of mutations, and their analysis in the context of the ATP synthase complex . Three classes of mutants will be considered in this review . (1) Cys substitutions have been used for structural analysis of subunit a, and its interactions with subunit c . (2) Functional residues have been identified by extensive mutagenesis . These studies have included the identification of second-site suppressors within subunit a . (3) Disruptive mutations include deletions at both termini, internal deletions, and single amino acid insertions . The results of these studies, in conjunction with information about subunits b and c, can be incorporated into a model for the mechanism of proton translocation in the Escherichia coli ATP synthase.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 417 - 27
Molecular models of the structural arrangement of subunits and the mechanism of proton translocation in the membrane domain of F(1)F(0) ATP synthase; Groth G; Subunit c of the proton-transporting ATP synthase of Escherichia coli forms an oligomeric complex in the membrane domain that functions in transmembrane proton conduction . The arrangement of subunit c monomers in this oligomeric complex was studied by scanning mutagenesis . On the basis of these studies and structural information on subunit c, different molecular models for the potential arrangement of monomers in the c-oligomer are discussed . Intersubunit contacts in the F(0) domain that have been analysed in the past by chemical modification and mutagenesis studies are summarised . Transient contacts of the c-oligomer with subunit a might play a crucial role in the mechanism of proton translocation . Schematic models presented by several authors that interpret proton transport in the F(0) domain by a relative rotation of the c-subunit oligomer against subunit a are reviewed against the background of the molecular models of the oligomer.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 404 - 16
The structure of the H(+)-ATP synthase from chloroplasts and its subcomplexes as revealed by electron microscopy; Bottcher B et al.; The electron microscopic data available on CF(0)F(1) and its subcomplexes, CF(0), CF(1), subunit III complex are collected and the CF(1) data are compared with the high resolution structure of MF(1) . The data are based on electron microscopic investigation of negatively stained isolated CF(1), CF(0)F(1) and subunit III complex . In addition, two-dimensional crystals of CF(0)F(1) and CF(0)F(1) reconstituted liposomes were investigated by cryo-electron microscopy . Progress in the interpretation of electron microscopic data from biological samples has been made with the introduction of image analysis . Multi-reference alignment and classification of images have led to the differentiation between different conformational states and to the detection of a second stalk . Recently, the calculation of three-dimensional maps from the class averages led to the understanding of the spatial organisation of the enzyme . Such three-dimensional maps give evidence of the existence of a third connection between the F(0) part and F(1) part.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 387 - 403
Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase; Fillingame RH et al.; F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme . Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1) . The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor . From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b . The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site . The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a . We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 374 - 86
Operation of the F(0) motor of the ATP synthase; Dimroth P; ATP, the universal carrier of cell energy is manufactured from ADP and phosphate by the enzyme ATP synthase using the energy stored in a transmembrane ion gradient . The two components of the ion gradient (DeltapH or DeltapNa(+)) and the electrical potential difference Deltapsi are thermodynamically but not kinetically equivalent . In contrast to accepted wisdom, the electrical component is kinetically indispensable not only for bacterial ATP synthases but also for that from chloroplasts . Recent biochemical studies with the Na(+)-translocating ATP synthase of Propionigenium modestum have given a good idea of the ion translocation pathway in the F(0) motor . Taken together with biophysical data, the operating principles of the motor have been delineated.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 364 - 73
The ATP synthase of Escherichia coli: structure and function of F(0) subunits; Deckers-Hebestreit G et al.; In this review we discuss recent work from our laboratory concerning the structure and/or function of the F(0) subunits of the proton-translocating ATP synthase of Escherichia coli . For the topology of subunit a a brief discussion gives (i) a detailed picture of the C-terminal two-thirds of the protein with four transmembrane helices and the C terminus exposed to the cytoplasm and (ii) an evaluation of the controversial results obtained for the localization of the N-terminal region of subunit a including its consequences on the number of transmembrane helices . The structure of membrane-bound subunit b has been determined by circular dichroism spectroscopy to be at least 75% alpha-helical . For this purpose a method was developed, which allows the determination of the structure composition of membrane proteins in proteoliposomes . Subunit b was purified to homogeneity by preparative SDS gel electrophoresis, precipitated with acetone, and redissolved in cholate-containing buffer, thereby retaining its native conformation as shown by functional coreconstitution with an ac subcomplex . Monoclonal antibodies, which have their epitopes located within the hydrophilic loop region of subunit c, and the F(1) part are bound simultaneously to the F(0) complex without an effect on the function of F(0), indicating that not all c subunits are involved in F(1) interaction . Consequences on the coupling mechanism between ATP synthesis/hydrolysis and proton translocation are discussed.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 356 - 63
The second stalk of Escherichia coli ATP synthase; Dunn SD et al.; Two stalks link the F(1) and F(0) sectors of ATP synthase . The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex . The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx . 125 A from the membrane . This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis . In vitro, b(2)delta is a highly extended complex held together by weak interactions . Recent work has identified the domains of b which are essential for dimerization and for interaction with delta . Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair . However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 289 - 99
Molecular mechanisms of rotational catalysis in the F(0)F(1) ATP synthase; Nakamoto RK et al.; Rotation of the F(0)F(1) ATP synthase gamma subunit drives each of the three catalytic sites through their reaction pathways . The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360 degrees rotation of the gamma subunit . Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor gamma subunit and the catalytic beta subunits . Amino acid substitutions, such as replacement of the conserved gammaMet-23 by Lys, cause altered interactions between gamma and beta subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions . The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient . Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.

J Biol Chem, 2000 Aug 18, 275(33), 25372 - 80
Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation; Clish CB et al.; The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution . 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4) . Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites . 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion . PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4) . With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4) . Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions . At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity . Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR) . In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking . These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation . Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.

J Biol Chem, 2000 Aug 18, 275(33), 25600 - 7
Refolding of target proteins from a "rigid" mutant chaperonin demonstrates a minimal mechanism of chaperonin binding and release; Mizobata T et al.; One of the most interesting facets of GroEL-facilitated protein folding lies in the fact that the requirement for a successful folding reaction of a given protein target depends upon the refolding conditions used . In this report, we utilize a mutant of GroEL (GroEL T89W) whose domain movements have been drastically restricted, producing a chaperonin that is incapable of utilizing the conventional cyclic mechanism of chaperonin action . This mutant was, however, still capable of improving the refolding yield of lactate dehydrogenase in the absence of both GroES and ATP hydrolysis . A very rapid interconversion of conformations was detected in the mutant immediately after ATP binding, and this interconversion was inferred to form part of the target release mechanism in this mutant . The possibility exists that some target proteins, although dependent on GroEL for improved refolding yields, are capable of refolding successfully by utilizing only portions of the entire mechanism provided by the chaperonins.

J Biomol Screen, 1999, 4(1), 3 - 7
High Throughput Scintillation Proximity Assay for the Identification of FKBP-12 Ligands; Graziani F et al.; A high throughput scintillation proximity assay (SPA) was developed to identify novel ligands of FKBP-12, an immunophilin with peptidyl prolyl isomerase (rotamase) activity . Recombinant histidine-tagged FKBP-12 was expressed in Escherichia coli, purified by metal ion affinity chromatography, and immobilized to SPA beads by an antibody that recognizes the histidine tag of the recombinant protein . Using 1 nM {3H} FK506, a well-known macrolid ligand of FKBP-12, specific binding was saturable and accounted for 95% of total binding . Analysis of saturation and homologous displacement isotherms indicated the existence of a single binding site with a Kd value of 1.6 nM . The specificity of {3H} FK506 binding was demonstrated in displacement experiments and showed that rapamycin, another macrolid, was as active as FK506 (IC50 of 3.5 and 3.2 nM, respectively), whereas GPI-1046, a prototype of small molecular compounds with neurotrophic properties and affinity for FKBP-type immunophilins, was more than 1000-fold less active . The high signal-to-noise ratio of 30, together with small standard deviations, makes this novel assay well suited for automated high throughput screening.

Adv Drug Deliv Rev, 1997 Jul 7, 26(2-3), 173 - 184
Gene directed enzyme prodrug therapy for cancer; Kerr DJ et al.; One strategy for gene therapy in malignant disease is gene directed enzyme prodrug therapy (GDEPT) . An exogenous enzyme gene is delivered to tumour cells . The enzyme, when expressed, can convert a non-toxic prodrug into a cytotoxic species that is capable of killing the cell in which it has been produced . The most frequently used systems are HSV thymidine kinase with ganciclovir and E . coli cytosine deaminase with 5-fluorocytosine . The bystander effect is of key importance to GDEPT: This describes the local spread of active species from cells that express the enzyme to kill adjacent, untransduced cells . The ultimate success of GDEPT will depend on the ability to achieve efficient gene delivery to and expression in target cells, whilst minimising expression in other tissues . A variety of techniques exist to achieve this goal, including loco-regional administration, manipulation of tumour blood supply and use of tumour-specific promoters to drive enzyme gene expression.

Carcinogenesis, 2000 Jun, 21(6), 1259 - 62
Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene; Baross-Francis A et al.; We reported previously that thymic lymphomas arising in mice lacking the DNA mismatch repair (MMR) gene, Msh2(-/-), exhibited striking elevations in the mutation frequency of a transgenic lacI reporter gene when compared with normal Msh2(-/-) tissues . To investigate whether hypermutation was a feature of all tumors arising in MMR-deficient mice, lacI transgene mutation frequencies were obtained from several different mouse tumors deficient for PMS2 and/or MSH2 . While lacI gene hypermutation was again clearly evident in Msh2 +/- ms2(-/-) and Msh2(-/-)Pms2(-/-) thymic lymphomas, three non-thymic MSH2-deficient tumors failed to show lacI gene mutation frequency elevations when compared with a normal tissue of MMR-deficient mice . The elevated mutation frequencies in the lymphoid tumors, and the finding of multiple clustered mutations in lacI genes rescued from these tumors, suggest that they are possibly generated by a lymphoma-specific hypermutational mechanism.

RNA, 2000 May, 6(5), 744 - 54
Puromycin-rRNA interaction sites at the peptidyl transferase center; Rodriguez-Fonseca C et al.; The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii . Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin . They include A2439, G2505, and G2553 for E . coli, and G2058, A2503, G2505, and G2553 for Hf . gibbonsii (using the E . coli numbering system) . Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E . coli 23S rRNA . In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E . coli ribosomes . Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E . coli 23S rRNA . These data strongly support the concept that puromycin, along with other peptidyl-transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region . This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs . In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.

RNA, 2000 May, 6(5), 680 - 6
Effects of anticodon 2'-O-methylations on tRNA codon recognition in an Escherichia coli cell-free translation; Satoh A et al.; The methylation of 2'-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules . Some tRNA species have a 2'-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex . However, no tRNA species have been found to have the modification at the second or third position of the anticodon . In the present study, we measured the effects of anticodon 2'-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay . The modification of C in the first position of the anticodon into 2'-O-methylcytidine increased the efficiency of reading the G-ending codon . On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity . Thus, 2'-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2'-O-methyl nucleosides are not found in these positions within natural tRNA species.

J Photochem Photobiol B, 2000 Feb, 54(2-3), 155 - 61
Non-coherent visible and infrared radiation increase survival to UV (254 nm) in Escherichia coli K12; Lage C et al.; Interactions between visible or infrared (IR) and ultraviolet (UV, 254 nm) radiation have been studied in E . coli . Pre-illumination with non-coherent monochromatic 446, 466, 570 and 685 nm radiation, as well as with polychromatic red and IR radiation at room temperature, leads to increased cell survival after a subsequent irradiation with UV light . In the thermic range of the spectrum (red and IR), IR but not red light pre-treatment is able to increase cell survival to a subsequent lethal heat (51 degrees C) challenge, suggesting that increased UV survival may be due to IR-induced heat-shock response . On the other hand, visible-light-induced resistance may be due to a different mechanism, possibly involved with unknown bacterial light receptors.

Parasitol Res, 2000 May, 86(5), 431 - 5
The gene encoding the metacyclogenesis-associated transcript Mat-1 is conserved in the genus Leishmania and shows a tendency to form dimers upon protein expression; Marin M et al.; The Leishmania infantum Mat-1 gene--recently described in L . major as a highly stage-specific, metacyclogenesis-associated transcript--has been cloned . The 420-bp Mat-1 coding region is conserved with respect to the L . major gene (82% sequence homology) . Analysis of the predicted amino-acid sequence reveals structural motifs showing homology with the class of leucine-zipper transcription factors . Southern-blot hybridization analysis suggests that Mat-1 is a low-copy-number gene, probably consisting of two gene copies . The recombinant Mat-1 protein expressed in fusion with the Escherichia coli maltose-binding protein shows a tendency to form dimers in the presence of the leucine-rich C-terminal domain . Bacteria expressing the Mat-1 open reading frame are highly growth-attenuated and tend to delete or modify the insert, which suggests that expression of Mat-1 is toxic for the bacteria.

Philos Trans R Soc Lond B Biol Sci, 2000 Apr 29, 355(1396), 491 - 501
Constraints on models for the flagellar rotary motor; Berg HC; Most bacteria that swim are propelled by flagellar filaments, each driven at its base by a rotary motor embedded in the cell wall and cytoplasmic membrane . A motor is about 45 nm in diameter and made up of about 20 different kinds of parts . It is assembled from the inside out . It is powered by a proton (or in some species, a sodium-ion) flux . It steps at least 400 times per revolution . At low speeds and high torques, about 1000 protons are required per revolution, speed is proportional to protonmotive force, and torque varies little with temperature or hydrogen isotope . At high speeds and low torques, torque increases with temperature and is sensitive to hydrogen isotope . At room temperature, torque varies remarkably little with speed from about -100 Hz (the present limit of measurement) to about 200 Hz, and then it declines rapidly reaching zero at about 300 Hz . These are facts that motor models should explain . None of the existing models for the flagellar rotary motor completely do so.

Nat Med, 2000 Jun, 6(6), 680 - 5
FcalphaRI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity; van Egmond M et al.; Despite the well-recognized involvement of immunoglobulin (Ig) A in mucosal immunity, the function of its receptor, FcalphaRI (CD89), is poorly understood . The ability of FcalphaRI to activate leukocytes seems to conflict with the proposed anti-inflammatory activity of secretory IgA . We show here that in a transgenic mouse model, inflammatory mediators induced expression of FcalphaRI on Kupffer cells, which enabled efficient phagocytosis in vivo of bacteria coated with serum IgA . Secretory IgA did not initiate phagocytosis . Therefore, interactions between serum IgA and FcalphaRI on Kupffer cells may provide a 'second line of defense' in mucosal immunity, by eliminating invasive bacteria entering through the portal circulation and thus preventing disease.

J Biol Chem, 2000 Aug 11, 275(32), 25000 - 7
Distinct membrane binding properties of N- and C-terminal domains of Escherichia coli SecA ATPase; Dapic V et al.; SecA is a motor protein that drives protein translocation at the Escherichia coli translocon . SecA membrane binding has been shown to occur with high affinity at SecYE and low affinity at anionic phospholipids . To dissect SecA-membrane interaction with reference to SecA structure, the membrane binding properties of N- and C-terminal SecA domains, denoted SecA-N664 and SecA-619C, respectively, were characterized . Remarkably, only SecA-N664 bound to the membrane with high affinity, whereas SecA-619C bound with low affinity in a nonsaturable manner through partitioning with phospholipids . Moreover, SecA-N664 and SecA-619C associated with each other to reconstitute wild type binding affinity . Corroborative results were also obtained from membrane binding competition and subcellular fractionation studies along with binding studies to membranes prepared from strains overproducing SecYE protein . Together, these findings indicate that the specific interaction of SecA with SecYE occurs through its N-terminal domain and that the C-terminal domain, although important in SecA membrane cycling at a later stage of translocation, appears to initially assist SecA membrane binding by interaction with phospholipids . These results provide the first evidence for distinct membrane binding characteristics of the two SecA primary domains and their importance for optimal binding activity, and they are significant for understanding SecA dynamics at the translocon.

Genetics, 2000 Jun, 155(2), 487 - 97
Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12; Sandler SJ; In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates . Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo . Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan . These determinations were made using a nonselective P1 transduction-based viability assay . Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality . Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case . Additionally, it was shown that the absence of the 3'-5' helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant . One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways . The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep . It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.

Nitric Oxide, 2000 Apr, 4(2), 112 - 22
Effects of short-term nitrogen monoxide inhalation on leukocyte adhesion molecules, generation of reactive oxygen species, and cytokine release in human blood; Opdahl H et al.; Increased nitrogen monoxide (NO) concentrations change leukocyte function under a multitude of experimental conditions . NO inhalation is an experimental treatment for lung failure and exposes leukocytes to increased NO concentrations during passage through the lungs . To investigate whether short-term NO inhalation induces lasting changes in the function of circulating human leukocytes, venous blood samples were drawn from eight healthy male volunteers before and at the end of a 35-min period of breathing 40 ppm NO in 30% O(2) . The leukocytes in the samples were subsequently analyzed for NO-induced changes in expression of cell surface molecules, generation of reactive oxygen species (ROS), and cytokine production by flow cytometry and ELISA techniques . The results were (1) NO inhalation changed neither the baseline nor the Escherichia coli lipopolysaccharide (LPS)-induced expression of the cell adhesion molecules CD11a, CD11b, CD11c, and CD62L (l-selectin) on neutrophilic granulocytes (PMN) or monocytes (Mo) . The expression of CD14 and HLA-DR was also unchanged . (2) The generation of ROS in response to activation with phorbol myristate acetate increased in PMN after NO inhalation; an increase in Mo did not reach significance . (3) Baseline and LPS-stimulated production of IL-1beta decreased after NO inhalation, while the LPS-stimulated production of TNF-alpha increased . No changes in IL-6 production were detected .

J Mol Biol, 2000 Jun 9, 299(3), 805 - 12
Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg . C; Gerk LP et al.; We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli . Wild-type tetrameric LacR denatures irreversibly at 53 degrees C . Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg . C without abolishing inducer binding at high and low temperatures . Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C . We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact . Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece . The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C . LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose . The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core .

J Mol Biol, 2000 Jun 9, 299(3), 757 - 70
Molten globule structure of equine beta-lactoglobulin probed by hydrogen exchange; Kobayashi T et al.; The molten globule structure of equine beta-lactoglobulin has been inferred from the hydrogen exchange protection of the backbone amide protons . In order to make it possible to measure the hydrogen exchange kinetics of the individual backbone amide protons, the uniformly (15)N-labeled recombinant protein was expressed in Escherichia coli and the NMR peak assignment was obtained for most of the backbone protons . The chemical shift and NOE results obtained under the condition where the protein assumes the native structure are fully consistent with the known secondary structure of bovine beta-lactoglobulin, indicating that the equine protein has a similar native conformation to that of the bovine protein . The hydrogen exchange rate of the individual backbone amide protons was measured under the conditions where the protein assumes the native and molten globule states . In the native state, strong protection was observed for the residues located in the eight (A to H) strands, which form a barrel structure, and residues of a major helix . In the molten globule state at acidic pH conditions, significant protection from the exchange has been observed for residues located in the A, F, G and H strands in the native structure . The pattern of protection is consistent with a native-like beta-sheet formation by these strands . The residues located in a major helix of the native structure are also protected, suggesting that this helix is formed in the molten globule and is packed against the sheet as in the native structure . These results indicate that a native-like subdomain is formed in the molten globule state of equine beta-lactoglobulin .

J Mol Biol, 2000 Jun 9, 299(3), 629 - 40
Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex; Malkov VA et al.; A fundamental problem in homologous recombination is how homology between DNAs is recognized . In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology . When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex . A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex . Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove . The structure implies that homology is recognized in the major groove of the duplex .

J Mol Biol, 2000 Jun 9, 299(3), 615 - 28
Initiation factor 3-induced structural changes in the 30 S ribosomal subunit and in complexes containing tRNA(f)(Met) and mRNA; Shapkina TG et al.; Initiation factor 3 (IF3) acts to switch the decoding preference of the small ribosomal subunit from elongator to initiator tRNA . The effects of IF3 on the 30 S ribosomal subunit and on the 30 S.mRNA . tRNA(f)(Met) complex were determined by UV-induced RNA crosslinking . Three intramolecular crosslinks in the 16 S rRNA (of the 14 that were monitored by gel electrophoresis) are affected by IF3 . These are the crosslinks between C1402 and C1501 within the decoding region, between C967xC1400 joining the end loop of a helix of 16 S rRNA domain III and the decoding region, and between U793 and G1517 joining the 790 end loop of 16 S rRNA domain II and the end loop of the terminal helix . These changes occur even in the 30 S.IF3 complex, indicating they are not mediated through tRNA(f)(Met) or mRNA . UV-induced crosslinks occur between 16 S rRNA position C1400 and tRNA(f)(Met) position U34, in tRNA(f)(Met) the nucleotide adjacent to the 5' anticodon nucleotide, and between 16 S rRNA position C1397 and the mRNA at positions +9 and +10 (where A of the initiator AUG codon is +1) . The presence of IF3 reduces both of these crosslinks by twofold and fourfold, respectively . The binding site for IF3 involves the 790 region, some other parts of the 16 S rRNA domain II and the terminal stem/loop region . These are located in the front bottom part of the platform structure in the 30 S subunit, a short distance from the decoding region . The changes that occur in the decoding region, even in the absence of mRNA and tRNA, may be induced by IF3 from a short distance or could be caused by the second IF3 structural domain .

Biotechnol Prog, 2000 May-Jun, 16(3), 385 - 90
Prolonging cell-free protein synthesis by selective reagent additions; Kim DM et al.; Factors causing the early cessation of protein synthesis have been studied in a cell-free system from Escherichia coli . We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ATP regeneration, and several amino acids are rapidly degraded during the cell-free protein synthesis reaction . The degradation of such compounds takes place even in the absence of protein synthesis . This degradation severely reduces the capacity for protein synthesis . The lost potency was completely recovered when the reaction mixture was supplied with additional PEP and amino acids . Of the 20 amino acids, only arginine, cysteine, and tryptophan were required to restore system activity . Through repeated additions of PEP, arginine, cysteine,and tryptophan, the duration of protein synthesis was greatly extended . In this fed-batch reaction, after a 2 h incubation, the level of cell-free synthesized chloramphenicol acetyl transferase (CAT) reached 350 microg/mL, which is 3.5 times the yield of the batch reaction . Addition of fresh magnesium further extended the protein synthesis . As a result, through coordinated additions of PEP, arginine, cysteine, tryptophan, and magnesium, the final concentration of cell-free synthesized CAT increased more than 4-fold compared to a batch reaction . SDS-PAGE analysis of such a fed-batch reaction produced an obvious band of CAT upon Coomassie Blue staining.

Protein Eng, 2000 May, 13(5), 369 - 76
Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay; Arai R et al.; A site-specific and efficient fluorolabeling of antibody variable regions with green fluorescent protein (GFP) variants and its application to an energy transfer-based homogeneous fluoroimmunoassay (open sandwich FIA) were attempted . Two chimeric proteins, Trx-V(H)-EBFP and Trx-V(L)-EGFP, consisting of V(H) and V(L) fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFP color variants, EBFP and EGFP, respectively, were designed to be expressed in cytoplasm of trxB - mutant Escherichia coli as fusions with thioredoxin from E.coli The mixture of two proteins could be purified with HEL-affinity chromatography, retaining sufficient intrinsic fluorescence and binding activity to HEL . A significant increase in fluorescence resonance energy transfer (FRET) dependent on HEL concentration was observed, indicating the reassociation of the V(H) and V(L) domains of these chimeric proteins due to co-existing antigen . With this open sandwich FIA, an HEL concentration of 1-100 microg/ml could be non-competitively determined . The assay could be performed in a microplate format and took only a few minutes to obtain a sufficient signal after simple mixing of the chimeric proteins with samples . This represents the first demonstration that the FRET between GFP variants is applicable to homogeneous immunoassay.

J Clin Microbiol, 2000 Jun, 38(6), 2162 - 9
Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O118 display three distinctive clonal groups of EHEC pathogens; Wieler LH et al.; A recent case report of a child infected with enterohemorrhagic Escherichia coli (EHEC) of serotype O118:H16 in Bavaria, in association with the isolation of a bovine O118 strain on the same farm (A . Weber, H . Klie, H . Richter, P . Gallien, M . Timm, and K . W . Perlberg, Berl . Muench . Tieraerztl . Wochenschr . 110:211-213, 1997), prompted us to investigate the relationship between bovine and human strains of serogroup O118 . A total of 29 human O118 E . coli strains from Europe (21), Canada (4), and Peru (4) were compared by virulence typing and macrorestriction analysis with 7 bovine O118 EHEC strains isolated in Bavaria . Twenty-five of the human strains were characterized as EHEC . By serotyping and determination of the virulence-associated factors Shiga toxin (stx1 stx2 stx2 variants), intimin (eae), and EHEC hemolysin (Hly(EHEC)), three distinctive groups of O118 human pathogens were identified . Most of the strains belonged to serotype O118:H16, displaying the virulence traits Stx1, intimin, Hly(EHEC), and EspP/PssA (group 1) . In addition, we identified strains of serotype O118:H12 (Stx2d only; group 2) and of serotype O118:H30 (Stx2 and intimin; group 3) . Macrorestriction analysis with BlnI and XbaI revealed that all strains with a single O118 serotype profile (O118:H12, O118:H16, and O118:H30) belonged to one clonal cluster, irrespective of their origin . Group 1 strains clustered in the same clonal group as the bovine O118:H16 strains . Moreover, four pairs of strains of different origins and indistinguishable by all other methods applied were identified as group 1 strains . Our data support the direct transmission of an EHEC O118:H16 strain from a calf to a 2-year-old boy in the above-mentioned case report . Since bovine and human O118:H16 strains represent the same clones, they must be considered zoonotic EHEC pathogens . In contrast, EHEC strains of serotypes O118:H12 and O118:H30 have been isolated only from humans, indicating a reservoir for certain human O118 EHEC strains other than bovines.

Science, 2000 Jun 2, 288(5471), 1643 - 7
Uninterrupted MCM2-7 function required for DNA replication fork progression; Labib K et al.; Little is known about the DNA helicases required for the elongation phase of eukaryotic chromosome replication . Minichromosome maintenance (MCM) protein complexes have DNA helicase activity but have only been functionally implicated in initiating DNA replication . Using an improved method for constructing conditional degron mutants, we show that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae . Like the Escherichia coli dnaB and SV40 T antigen helicases, therefore, the MCM complex is loaded at origins before initiation and is essential for elongation . Restricting MCM loading to the G(1) phase ensures that initiation and elongation occur just once per cell cycle.

Crit Care Med, 2000 May, 28(5), 1526 - 33
Coupled plasma filtration-adsorption in a rabbit model of endotoxic shock; Tetta C et al.; OBJECTIVE: To test the hypothesis that nonselective adsorption by a hydrophobic resin of cytokines and other proinflammatory mediators could improve 72-hr survival in a rabbit model of endotoxic shock . DESIGN: Prospective, randomized, controlled animal trial . SETTING: Animal care facility at a research institution . SUBJECTS: A total of 109 New Zealand white male rabbits . INTERVENTIONS: Anesthetized rabbits were cannulated with indwelling femoral arterial and venous lines . Septic shock was induced by a single intravenous injection of Escherichia coli lipopolysaccharide . The dose was experimentally assessed in 40 rabbits receiving 1.0, 0.5, 0.1, and 0.05 mg/kg body weight to determine LD80 at 72 hrs . Extracorporeal circulation consisted of plasma filtration coupled with passage of the plasma filtrate through a hydrophobic sorbent and reinfusion into the venous line . The extracorporeal treatment lasted for 3 hrs . Rabbits injected with endotoxin (0.05 mg/kg) were submitted to plasma filtration with (19 rabbits) or without (20 rabbits) sorbent adsorption . As controls, rabbits injected with vehicle alone were treated with plasma filtration (ten rabbits) or without (ten rabbits) sorbent adsorption . Ten rabbits were monitored under anesthesia to determine basal survival . MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of endotoxin, bioactive tumor necrosis factor, resin-adsorbed platelet-activating factor, mean arterial pressure, base excess, and white cell count were assessed and a global severity score was established . At 72 hrs, cumulative survival was significantly (p = .0041) improved in septic rabbits treated with coupled plasma filtration-adsorption . Circulating tumor necrosis factor bioactivity remained similar in control and treated rabbits . Biologically significant amounts of platelet activating factor were eluted from the sorbent during the entire treatment time . The severity score inversely correlated with survival (p < .001) . CONCLUSIONS: Coupled plasma filtration-adsorption improved survival in a rabbit model of endotoxic shock . Coupled plasma filtration-adsorption may be an extracorporeal treatment capable of removing structurally different inflammatory mediators associated with sepsis.

Crit Care Med, 2000 May, 28(5), 1515 - 21
Endothelin-1 impairs neutrophil respiratory burst and elimination of Escherichia coli in rabbits; Heller A et al.; OBJECTIVE: During systemic inflammation, elevated levels of endothelin (ET)-1 have been reported . The aim of this study was to investigate the effects of ET-1 on neutrophil (PMN) respiratory burst, phagocytosis, and elimination of Escherichia coli from blood and tissues . DESIGN: Prospective, randomized, controlled trial . SETTING: Experimental laboratory in a university hospital . SUBJECTS: A total of 18 female chinchilla rabbits . INTERVENTIONS: To quantify the clearance process, defined numbers (10(8) colony-forming units) of E . coli were injected intravenously into anesthetized rabbits, 60 mins after onset of continuous 0.2 microg/kg/min ET-1 administration (n = 9) and after saline infusion (control group, n = 9), respectively . To evaluate potential effects of ET-1 on bacterial elimination and killing, blood clearance of E . coli and colonization of different organs were investigated . MEASUREMENTS: Variables monitored were neutrophil respiratory burst and phagocytosis activity, rates of bacterial elimination from the blood, arterial blood pressure, blood gases, serum lactate concentrations, and nitrite and nitrate levels . The animals were killed 3 hrs after bacterial injection and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts . MAIN RESULTS: Compared with the control group, ET-1 significantly impaired PMN respiratory burst (p < .05) and prolonged elimination of injected E . coli from the blood (p < .01), whereas phagocytosis functions remained unaltered . The reduced PMN burst activity after ET-1 was associated with a higher bacterial colonization of all organs (lung, p < .01; spleen, p < .05) . Endothelin-1 induced increases in mean arterial pressure (p < .01) and serum lactate concentrations, whereas nitrite and nitrate levels remained unaltered . CONCLUSION: Endothelin-1 impairs respiratory burst and bacterial clearance from the blood and tissue . Thus, elevated levels of ET-1 during sepsis could induce organ hypoperfusion and cause disturbances in immune functions, increasing the risk of bacterial infections.

Crit Care Med, 2000 May, 28(5), 1439 - 44
Endotoxin induces a dose-dependent myocardial cross-tolerance to ischemia-reperfusion injury; Neviere RR et al.; OBJECTIVE: To test whether or not endotoxin induces a dose-dependent reduction of myocardial contractile dysfunction after a standardized period of myocardial ischemia and reperfusion and whether nitric oxide is involved in this form of myocardial protection . DESIGN: Prospective, randomized, controlled animal study . SETTING: University research laboratory . SUBJECTS: Twenty-five male Sprague-Dawley rats . INTERVENTIONS: After anesthesia, the left carotid artery was cannulated under sterile conditions and animals were allowed to recover from surgery for 12 hrs . Sterile saline or increasing doses (2.5, 5, or 10 mg/kg body weight) of endotoxin (Escherichia coli O26:B6; Sigma, Mississauga, Ontario, Canada) were given intravenously (1 mL over 5 mins) . In some rats, diaspirin-crosslinked hemoglobin (200 mg/kg) was infused 6 hrs and 60 min before endotoxin infusion (10 mg/kg) . Hearts were rapidly excised for retrograde perfusion through the ascending aorta (Langendorff apparatus) 6 hrs later . After baseline data collection, hearts were subjected to global ischemia (30 mins, 37 degrees C {98.6 degrees F}), followed by 30 mins of reperfusion . MEASUREMENTS AND MAIN RESULTS: Physiologic variables were recorded 6 hrs after saline and endotoxin infusion . Baseline myocardial systolic contractility and diastolic compliance were assessed, respectively, by left ventricular developed pressure (LVDP) and left ventricular (LV) volume-preload relationships . After 30 min of reperfusion, LVDP recovery and left ventricular end-diastolic pressure were measured . Endotoxin induced LV systolic contractile depression, irrespective of the dose of endotoxin administered . LV diastolic dysfunction varied between different doses of endotoxin administered . On reperfusion, endotoxin produced a dose-dependent improvement of postischemic LVDP recovery: 30+/-6% in sham, 78+/-9% in 2.5 mg/kg, 93+/-8% in 5 mg/kg, and 107+/-10% in 10 mg/kg endotoxin heart . In rats treated with 10 mg/kg endotoxin, diaspirin-crosslinked hemoglobin pretreatment abrogated endotoxin-induced postischemic LVDP recovery improvement (105+/-10% vs . 43+/-7%, p = .01) . CONCLUSION: Sublethal doses of endotoxin induce in a dose-dependent manner a delayed form of myocardial protection against ischemia . Although free-cell hemoglobin solution abrogates this endotoxin-induced cross-tolerance, we propose that possible mechanisms involved in this form of myocardial protection include nitric oxide pathway activation.

J Anim Sci, 2000 May, 78(5), 1310 - 2
Technical note: recombinant LHRH fusion protein suppresses estrus in heifers; Sosa JM et al.; A recombinant luteinizing hormone-releasing hormone (LHRH) fusion protein was evaluated for its effectiveness in suppression of estrus in heifers . Eight heifers were randomly assigned to two equal treatment groups . Treatments consisted of recombinant ovalbumin-LHRH-7 or recombinant ovalbumin (control) . This recombinant chimeric fusion protein consisted of ovalbumin with seven LHRH peptides (ovalbumin-LHRH-7) . The plasmid for this protein was expressed in E . coli and was collected and purified as an insoluble protein . One milligram of the respective proteins was suspended in 2 mL of Z-Max adjuvant and administered by intramammary injection three times at 7-wk intervals . Luteinizing hormone-releasing hormone antibody binding was elevated in heifers treated with ovalbumin-LHRH-7 compared to ovalbumin-treated heifers (P < .05) . Serum progesterone concentrations (< 1 ng/mL) indicate that the estrous cycle of the four heifers treated with ovalbumin-LHRH-7 was suppressed for a time period ranging from 60 to 238 d, which was different from control heifers (P < .01) . Serum progesterone for the control heifers continued to exhibit cyclic profiles over the experimental period . This preliminary study in heifers demonstrated that a chimeric LHRH fusion protein induced elevated concentrations of circulating LHRH antibodies that suppressed estrus for an average of 122 +/- 41 d.

Ann Hematol, 2000 Apr, 79(4), 187 - 97
Oxidative burst measurement in patients treated with cytostatics: influence of G-CSF and role as a prognostic factor; Volk J et al.; The ability to generate reactive oxygen species, the so-called oxidative burst, is essential for neutrophils to kill infectious micro-organisms . Flow cytometry was used to study oxidative burst prior to, during, and after cytostatic therapy . Seven patients were treated according to the DexaBEAM regimen with 12 cycles monitored . Four patients were treated according to the B-NHL regimen in which nine cycles were monitored . Ten healthy volunteers were chosen as a control group without any treatment . Neutrophils were collected from heparinized peripheral blood and were stimulated by phorbol-12-myristate-13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), and Escherichia coli . The oxidative burst was estimated by the amount of nonfluorescent dihydrorhodamine 123 converted to green fluorescent rhodamine 123 . Measurements were done daily . The FMLP-induced burst was enhanced in patients before therapy as compared with the control group, whereas PMA-induced burst was decreased slightly . E . coli-, FMLP-, and PMA-induced oxidative burst decreased in both groups during cytostatic therapy . E . coli-induced burst increased again within 2 days of G-CSF treatment in vivo . FMLP-induced burst increased in the B-NHL group but decreased in the DexaBEAM group . In patients who have recovered from leukopenia the oxidative burst is still partly suppressed . PMA-induced oxidative burst measured at the start of therapy correlates with infectious complications . Thus, PMA-induced burst may be used as a simple method for evaluating the individual risk of infections during therapy . The results demonstrate the modulating effect of cytostatic drugs on the oxidative burst and may explain why some patients suffer from severe bacterial infections although the total number of granulocytes is normal.

Anticancer Drug Des, 1999 Dec, 14(6), 507 - 15
Intensely cytotoxic anthracycline prodrugs: galactosides; Bakina E et al.; We have reported the synthesis of a series of anthracycline analog prodrugs that give rise to intensely cytotoxic metabolites in the presence of carboxylate esterases and beta-glucuronidases . We now report structurally related prodrugs that are converted to similar potent metabolites in the presence of beta-galactosidases . The prototypical compound, N-{(4"RS)-4"-ethoxy-4"(1'"-O-beta-D-galactopyranosyl)butyl}daunorubicin, 8a, was prepared by reductive condensation of daunomycin with 1-O-{(1'RS)-1'-ethoxy-4'-oxobutyl}-2, 3, 4, 6-tetra-O-acetyl-beta-D-galactopyranoside in the presence of sodium cyanoborohydride, followed by deacetylation of the galactoside moiety with sodium methoxide . A related prodrug (8b) with enhanced lipophilicity (the 4'-hexoxy analog of 8a) and 8c (the propyldaunomycin analog of 8a) were prepared for comparative studies . 8a and 8b were isolated after chromatography on silica as a mixture of 4'R and 4'S diastereomers; 8c, on the other hand, was resolved into its component 3' diastereomers, 8c(R) and 8c(S) . 8a, 8c(R) and 8c(S) showed no evidence of decomposition when incubated at 37 degrees C in 0.05 M phosphate buffer, pH 7.4, for 2 weeks; 8b, under the same conditions, was degraded with a half-life of 49 h . In the presence of two units of Escherichia coli beta-galactosidase per pmol of substrate, the half-lives of 8a, 8b, 8c(R) and 8c(S) were 1.98, 1.06, 3.5 and 2.4 h, respectively . HPLC analysis of the incubation mixtures showed that 8a and 8b gave rise to a single, chromatographically identical metabolite . 8c(R) and 8c(S) also gave rise to a single, identical metabolite . 8a and 8b were nearly one million-fold more toxic to human A375 melanoma cells in culture in the presence of E . coli beta-galactosidase than in the absence of the enzyme . The activation products of 8c(R) and 8c(S) were approximately 1000-fold less potent . These beta-galactoside prodrugs have chemotherapeutic potential for use in conjunction with tissue-targeting strategies such as antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).

Anticancer Drug Des, 1999 Dec, 14(6), 461 - 72
Virus-directed enzyme prodrug therapy using CB1954; Grove JI et al.; The virus-directed enzyme prodrug therapy (VDEPT) anti-cancer 'gene therapy' strategy relies on the use of viral vectors for the efficient delivery to tumour cells of a 'suicide gene' encoding an enzyme which converts a non-toxic prodrug to a cytotoxic agent . The prodrug 5-(aziridin-1-yl)-2,4 dinitrobenzamide, CB1954, has been proposed for use in enzyme-prodrug gene therapy systems with the Escherichia coli enzyme nitroreductase (Ntr) . Ntr converts CB1954 to 2- and 4-hydroxylamino derivatives, whereupon the non-enzymatic reaction of the 4-hydroxylamino derivative with cellular thio- esters generates a potent cytotoxic bifunctional alkylating agent capable of cross-linking DNA . Ntr delivery has been achieved in vitro using retroviral and adenoviral vectors and confirmed by immunocytochemical demonstration of Ntr expression . The Ntr-expressing cells have been shown to be sensitized to CB1954 by up to 2000-fold . The Ntr-CB1954 system shows effective bystander killing in mixed populations of Ntr-expressing and non-expressing cells treated with CB1954 . The efficacy of this enzyme-prodrug approach in model systems compared with other VDEPT approaches demonstrates the feasibility and future promise of this gene therapy strategy.

J Biol Chem, 2000 Aug 25, 275(34), 26187 - 95
Identification of a region of Escherichia coli DnaB required for functional interaction with DnaG at the replication fork; Chang P et al.; The fundamental activities of the replicative primosomes of Escherichia coli are provided by DnaB, the replication fork DNA helicase, and DnaG, the Okazaki fragment primase . As we have demonstrated previously, DnaG is recruited to the replication fork via a transient protein-protein interaction with DnaB . Here, using site-directed amino acid mutagenesis, we have defined the region on DnaB required for this protein-protein interaction . Mutations in this region of DnaB affect the DnaB-DnaG interaction during both general priming-directed and phiX174 complementary strand DNA synthesis, as well as at replication forks reconstituted in rolling circle DNA replication reactions . The behavior of the purified mutant DnaB proteins in the various replication systems suggests that access to the DnaG binding pocket on DnaB may be restricted at the replication fork.

Gastroenterology, 2000 Jun, 118(6), 1051 - 60
Identification of transport abnormalities in duodenal mucosa and duodenal enterocytes from patients with cystic fibrosis; Pratha VS et al.; BACKGROUND & AIMS: The duodenum is a cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelium with high bicarbonate secretory capacity . We aimed to define the role of CFTR in human duodenal epithelial bicarbonate secretion in normal (NL) subjects and patients with cystic fibrosis (CF) . METHODS: Endoscopic biopsy specimens of the duodenal bulb were obtained from 9 CF patients and 16 volunteers . Tissues were mounted in modified Ussing chambers . Bicarbonate secretion and short-circuit current (Isc) were quantitated under basal conditions and in response to dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP), carbachol, and the heat-stable toxin of Escherichia coli (STa) . Duodenocytes were also isolated and loaded with the pH-sensitive fluoroprobe BCECF/AM, and intracellular pH (pH(i)) was measured at rest and after intracellular acidification and alkalinization . RESULTS: Basal HCO(3)(-) secretion and Isc were significantly lower in the CF vs . NL duodenal mucosa . In contrast to NL, db-cAMP failed to alter either HCO(3)(-) or Isc in CF tissues . However, in CF, carbachol resulted in an electroneutral HCO(3)(-) secretion, whereas STa induced electrogenic HCO(3)(-) secretion that was similar to NL . In CF and NL duodenocytes, basal pH(i) and recovery from an acid load were comparable, but pH(i) recovery after an alkaline load in CF duodenocytes was Cl(-) dependent, whereas in NL duodenocytes it was Cl(-) independent . CONCLUSIONS: These findings implicate CFTR in NL duodenal alkaline transport and its absence in CF . Although duodenal bicarbonate secretion is impaired in CF tissues, alternate pathway(s) likely exist that can be activated by carbachol and STa.

Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 596 - 602
Unusual substrate specificity of a chimeric hypoxanthine-guanine phosphoribosyltransferase containing segments from the Plasmodium falciparum and human enzymes; Sujay Subbayya IN et al.; Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the phosphoribosylation of hypoxanthine and guanine by transferring the phosphoribosyl moiety from phosphoribosylpyrophosphate (PRPP) on to N9 in the purine base, resulting in the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) . Xanthine is an additional substrate for the Plasmodium falciparum HGXPRT . Our aim has been to elucidate structural features in HGPRT that govern substrate specificity . We have addressed this problem by engineering chimeric HGPRTs, which contain segments from both the parasite and human enzymes . Four chimeric enzymes were engineered (DS1-DS4), of which the chimeric enzyme, DS1, in which the first 49 residues of human HGPRT were replaced with the corresponding residues from the P . falciparum enzyme, exhibited additional specificity for xanthine . None of the switched residues forms a part of the purine or PRPP binding region in the available crystal structures of HG(X)PRTs . Our data on the chimeric enzyme DS1 provide the first evidence that the N-terminal approximately 50 amino acids, although not proximal to the active site in the crystal structure, can in fact modulate substrate specificity . DS1 exhibits a reduced k(cat) for hypoxanthine and guanine, while its K(m) for these oxopurine bases remains largely unchanged . Its specific activity for xanthine is comparable with hypoxanthine but five times more than that for guanine .

Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 571 - 5
Escherichia coli genome is composed of two distinct types of nucleotide sequences; Haring D et al.; We calculated correlations of the nucleotide distributions along the E . coli genome . Subsequent cluster analysis of the correlation distributions showed that the genome was composed of two qualitatively different types of nucleotide sequences . The first type exhibited strong correlations of the genomic distributions of A with T and G with C, and high anticorrelations of A with C and G with T . In contrast, the second type was characterized by weak or negligible correlations typical of randomized sequences . Both types of sequences were almost equally abundant in the E . coli genome and their length varied from several hundred nucleotides to about 70 kilobases . They were not disjunct with respect to their (G + C) content but the high correlations and anticorrelations were rather characteristic for (A + T)-rich genomic segments . We offer possible explanations of the mosaic structure of the E . coli genome .

Protein Expr Purif, 2000 Jun, 19(1), 212 - 8
Functional and immunological analysis of recombinant mouse H- and L-ferritins from Escherichia coli; Santambrogio P et al.; The production and characterization of recombinant mouse H- and L-ferritin chains from Escherichia coli are described . The proteins were efficiently expressed and purified with yields of 7-40 mg per liter of cell culture . They had the expected molecular mass and showed a physical stability analogous to that of the corresponding human ferritins . Mouse H- and L-ferritins had a very similar mobility on denaturing SDS-PAGE, but could be readily separated on nondenaturing PAGE because of the distinct slow mobility of mouse L-ferritin . Direct comparative experiments showed that mouse and human H-ferritins had the same iron incorporation activity, whereas mouse L-ferritin incorporated iron less efficiently than human L-ferritin . The difference was attributed to the substitution of a residue exposed on the cavity surface (Glu140 --> Lys) in mouse L-ferritin, a hypothesis confirmed by the finding that the mouse L-ferritin mutant Lys140-Glu incorporated iron as efficiently as human L-ferritin . Rabbit antisera elicited by the recombinant mouse ferritins were specific for the H- and L-chains and did not cross-react with the human ferritins . The antibodies and the derived specific ELISA assays allow the determination of H- and L-ferritins in mouse tissues .

Protein Expr Purif, 2000 Jun, 19(1), 188 - 96
Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli; Luna-Chavez C et al.; Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II . The E . coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex . The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid . The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography . The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme . Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant . The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A . The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources .

Protein Expr Purif, 2000 Jun, 19(1), 139 - 47
Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes; Teske JG et al.; A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented . Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence . These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive . The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry . Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium . All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme . This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved . Two methods for assembling the holoenzyme are described . In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E . coli . A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants .

Protein Expr Purif, 2000 Jun, 19(1), 107 - 12
Cloning, expression, and purification of the His(6)-tagged thermostable beta-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme; Daabrowski S et al.; In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid . The nucleotide sequence revealed that beta-galactosidase of P . woesei consists of 510 amino acids and has a molecular weight of 59, 056 kDa (GenBank Accession No . AF043283) . It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus . We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S . D $$;abrowski, J . Maciunska, and J . Synowiecki, 1998, Mol . Biotechnol . 10, 217-222) . In this study we presented a new expression system for producing P . woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase) . The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography . The enzyme was purified by heat treatment (to denature E . coli proteins), followed by metal-affinity chromatography on Ni(2+)-TED-Sepharose columns . The enzyme was characterized and displayed high activity and thermostability . This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase .

Protein Expr Purif, 2000 Jun, 19(1), 53 - 6
The preparation of apo-Cu,Zn superoxide dismutase by ion-exchange chromatography on iminodiacetic acid-sepharose; Sutter B et al.; The superoxide dismutases (EC 1.15.1.1) are a family of enzymes that catalyze the dismutation of superoxide radical anion to dioxygen and hydrogen peroxide . The active site contains a critical metal ion such as manganese, iron, or copper . The copper-containing protein also has one zinc ion bound per subunit . The standard method used to remove the metal ions from Cu,Zn superoxide dismutase has been to exhaustively dialyze the protein against chelating agents at low pH . We have developed a new method where the protein is bound to ion-exchange medium based on iminodiacetic acid immobilized on Sepharose . The bound protein is treated with a buffer containing edta at pH 3.5 to remove metal ions; the buffer is then exchanged for acetate buffer to remove edta, after which the protein is eluted by a salt gradient . An advantage of this method is that a single chromatography step is sufficient to produce apo protein . Results are shown for both human and bovine dimeric Cu,Zn superoxide dismutase and the monomeric Escherichia coli Cu,Zn superoxide dismutase . In every case, the metals were removed efficiently .

Protein Expr Purif, 2000 Jun, 19(1), 41 - 7
Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease; Selistre-de-Araujo HS et al.; Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis . Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen IV, laminin, and fibronectin . A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity that could be very helpful in thrombosis therapy . We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library . The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells . Culture of the transformants at either 37 or 20 degrees C led to the overexpression of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction . The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharose column followed by gel filtration chromatography, both under denaturing conditions . After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis . The refolded recombinant protein was active in fibrin-agarose plates . The purified protein achieved a conformation similar to that of the native enzyme as judged by circular dichroism analysis .

Protein Expr Purif, 2000 Jun, 19(1), 22 - 9
Expression and purification of recombinant human indoleamine 2, 3-dioxygenase; Littlejohn TK et al.; Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases . The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography . The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract . When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein . The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme . This was as expected from the addition of the hexahistidyl tag . Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan . The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity . The recombinant protein can therefore be used to investigate properties of the native enzyme . This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression .

Protein Expr Purif, 2000 Jun, 19(1), 48 - 52
Overexpression and purification of fructose-1-phosphate kinase from Escherichia coli: application to the assay of fructose 1-phosphate; Veiga-da-Cunha M et al.; Fructose 1-phosphate is a metabolite that plays a regulatory role in liver and is best measured using an assay based on its conversion to fructose 1,6-bisphosphate by a bacterial fructose-1-phosphate kinase (Fru1PK) . The open reading frame encoding Escherichia coli Fru1PK has been introduced in an expression plasmid (pET3a) based on the T7 promoter-driven system, which was used to overexpress the enzyme . The conditions for the production of soluble Fru1PK were optimized . The purification procedure used involved ammonium sulfate precipitation and chromatography on DEAE-Sepharose and was aimed mostly at stabilizing the enzyme and at freeing Fru1PK from bacterial contaminants that could interfere in the fructose 1-phosphate assay . From a 1-liter culture, more than 50 mg protein is obtained . This preparation can be used in an enzymatic assay that measures specifically fructose 1-phosphate in tissue extracts .

J Biochem (Tokyo), 2000 Jun, 127(6), 1095 - 102
The effect of single residue substitutions of serine-283 on the strength of head-to-tail interaction and actin binding properties of rabbit skeletal muscle alpha-tropomyosin; Sano K et al.; Vertebrate skeletal muscle alpha-tropomyosin polymerizes in a head-to-tail manner and binds cooperatively to actin . It has been postulated that the cooperative actin binding is governed by the strength of the head-to-tail interaction . In order to know the relationship between the head-to-tail affinity and actin binding, we studied the properties of tropomyosin variants with single residue substitutions at serine-283, the penultimate residue at the carboxyl terminus that is involved in the head-to-tail interaction . It has been shown that the phosphorylation of serine-283 strengthens the head-to-tail interaction . Viscometry was employed to compare the head-to-tail affinity of tropomyosin variants . Variant S283E showed higher viscosity whereas variant S283K showed lower viscosity compared with the wild type non-phosphorylated alpha-tropomyosin . The results confirm the idea that the interaction is sensitive to the ionic properties of residue 283 . The strength of the head-to-tail interaction was assessed directly by sedimentation equilibrium using two pairs of tropomyosin variants designed so that only dimeric interactions were allowed within each pair . From one pair of variants with serine-283, the association constant was determined to be 2.6 x 10(4) M(-1) (SD =1.0 x 10(4)), whereas for the second pair with glutamate-283, the affinity was 3.9 x 10(4) M(-1) (SD =1.6 x 10(4)), slightly stronger than the former, consistent with the results of viscometry . The results indicate that the head-to-tail association is weak as previously implicated from light scattering measurements . Cosedimentation was employed to measure the cooperative actin binding of tropomyosin variants . Although previous results indicated the phosphorylation has no significant influence on the actin affinity, variant S283E shows a lower affinity compared with the control . Variants S283K and S283A show even lower affinities to actin, although these species bind to actin more cooperatively than does variant S283E . The results indicate that the affinity of the head-to-tail interaction between adjacent tropomyosin molecules is weak, and is substantially influenced by an extra charge at residue 283 . On the other hand, the interaction with actin, the affinity and the cooperativity in actin binding, is dependent on amino acid residues at 283 and is not simply correlated with the strength of the head-to-tail interaction between Tm molecules in solution.

J Biochem (Tokyo), 2000 Jun, 127(6), 1071 - 9
Characterization of a mutant form of SecA that alleviates a SecY defect at low temperature and shows a synthetic defect with SecY alteration at high temperature; Nakatogawa H et al.; The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane . We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42 degrees C . The overproduced SecA81 aggregated in vivo at temperatures above 37 degrees C . Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30 degrees C, while it was totally inactive at 42 degrees C . The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421-575 . This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein . In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42 degrees C . While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42 degrees C . The inactivated SecA81 molecules seemed to jam the translocase since it interfered with translocase functions at 42 degrees C . Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination . The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY.

J Biochem (Tokyo), 2000 Jun, 127(6), 1065 - 70
Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world; Kashiyama T et al.; In characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables . The speed of this sliding movement is the fastest among many molecular motors known so far . We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin . The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive alpha-helical coiled-coil domain, and a C-terminal globular domain . Phylogenetic analysis suggested that this myosin belongs to class XI.

J Biochem (Tokyo), 2000 Jun, 127(6), 1053 - 6
19F NMR investigation of F(1)-ATPase of Escherichia coli using fluorotryptophan labeling; Lee HW et al.; Growth of Escherichia coli in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, has permitted the production of proton-dislocating ATPase that is specifically labeled with 5-fluorotryptophan . Five sets of (19)F resonances could be assigned to each tryptophan residue by lauryldimethylamine oxide and carboxypeptidase treatment . On labeling with 4-chloro-7-nitro-benzofurazan, the label attached to b155Lys, which is known to be in the catalytic site, which caused one of the residues, b108Trp, to become nonequivalent . (19)F NMR spectroscopic investigation of internally fluorotryptophan-labeled F(1)-ATPase will provide valuable information about the asymmetric nature of F(1)-ATPase and the conformational changes induced by ligand binding.

J Biochem (Tokyo), 2000 Jun, 127(6), 955 - 63
Expression, purification, and characterization of blasticidin S deaminase (BSD) from Aspergillus terreus: the role of catalytic zinc in enzyme structure; Kimura M et al.; We established an efficient overproduction-purification system for blasticidin S deaminase (BSD) using the cDNA cloned from Aspergillus terreus . The estimated molecular mass of the purified enzyme indicated BSD was a tetramer . This tetrameric form was very resistant to denaturation by SDS and showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much slower (as a single band of 36 kDa) in its active conformation than its completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS was not performed before electrophoresis . As predicted from the presence of the catalytic zinc-coordinating sequence motif conserved in the cytosine nucleoside/nucleotide deaminase family, BSD also contained one zinc per deaminase subunit . However, the predicted catalytic function appeared not to be the only role of this zinc in the enzyme . First, titration of the zinc-chelating -SH groups with p-hydroxymercuriphenylsulfonate led to dissociation of the BSD tetramer into unstable monomers or dimers . Second, depletion of zinc on reconstitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) resulted in improper folding of the polypeptide . These results suggest that zinc also plays a structural role in maintenance of the protein structure . When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E56D, E56Q, C91A, C91S, and C91H) showed any detectable activity, as judged with the spectrophotometric assay . Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme . These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.

J Biochem (Tokyo), 2000 Jun, 127(6), 945 - 53
Backbone dynamics of the c-Myb DNA-binding domain complexed with a specific DNA; Sasaki M et al.; The DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3 . Each repeat contains three helices . The minimal DNA-binding domain is an R2R3 fragment . Here, we have examined the backbone dynamics of R2R3 in its DNA-bound form by NMR . Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible . In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2 . Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements . Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions . These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged . In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form . Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.

Pediatr Res, 2000 Jun, 47(6), 830 - 3
2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism; Gibson KM et al.; An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcamitine (2-MBC) in physiologic fluids . In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase) . Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli . Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance . These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.

Pediatr Res, 2000 Jun, 47(6), 736 - 42
Intrauterine infection induces programmed cell death in rabbit periventricular white matter; Debillon T et al.; An association between chorioamnionitis and periventricular leukomalacia has been reported in human preterm infants . However, whether this link is causal has not been convincingly established, and the underlying molecular mechanisms remain unclear . The objective of this study was to establish a reproducible model of cerebral white matter disease in preterm rabbits after intrauterine infection . Escherichia coli was inoculated into both uterine horns of laparotomized pregnant rabbits when gestation was 80% complete . The fetuses were delivered by cesarean section and killed 12, 24, or 48 h after the inoculation . Programmed cell death in the white matter was evaluated by hematoxylin-eosin-saffron staining and in situ fragmented DNA labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) . In a first group of 14 pregnant rabbits not treated with antibiotics, all fetuses delivered 48 h after inoculation were stillborn, whereas fetuses extracted 12 or 24 h after inoculation were alive . No significant cell death was detected in the live fetuses compared with the control noninfected rabbits . In a second group of five pregnant rabbits treated with ceftriaxone initiated 24 h after the inoculation and continued until cesarean section was performed 48 h after inoculation, 13 fetuses were alive, but all showed evidence of extensive programmed cell death in the white matter by hematoxylin-eosin-saffron staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling . White matter damage became histologically detectable only 48 h after inoculation . Three of the 13 brains displayed periventricular white matter cysts mimicking human cystic periventricular leukomalacia . The high reproducibility of white matter damage in our model should permit further studies aimed at unraveling the molecular mechanisms of periventricular leukomalacia.

Microbiology, 2000 May, 146 ( Pt 5), 1157 - 62
Isolation of a novel insertion sequence from Mycobacterium fortuitum using a trap vector based on inactivation of a lacZ reporter gene; Waskar M et al.; An insertion sequence of Mycobacterium fortuitum has been isolated using a trap vector following insertion in and inactivation of the lacZ reporter gene . The trap vector is a temperature-sensitive (ts) Escherichia coli-mycobacterium shuttle plasmid, pCD4, which contains ts oriM, the kanamycin-resistance gene as a selection marker and a lacZ expression cassette . The ts mutation present in pCD4 functions in mycobacteria and enables screening for transposable elements from the mycobacterial genome that disrupt the lacZ gene by screening for white colonies on X-Gal plates in both mycobacterial as well as E . coli hosts . The vector was used to isolate a novel 1.653 kb insertion sequence from M . fortuitum named IS219 . IS219 duplicated host DNA at the target site, had inverted repeats at its ends and contained two ORFs on one strand . One of the predicted proteins showed homology to a putative transposase from Acetobacter pasteurianus . IS219 was present in two copies in the genome of M . fortuitum . The trap vector appears to be useful in trapping insertion sequences from different mycobacteria by screening for the disrupted LacZ phenotype.

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