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Biochim Biophys Acta, 1998 Jun 29, 1385(2), 221 - 8 Activation of thiamin diphosphate in enzymes; Hubner G et al.; Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy . The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated . The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate . The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate. Nature, 1998 Jun 25, 393(6687), 812 - 7 Structure of a heparin-linked biologically active dimer of fibroblast growth factor; DiGabriele AD et al.; The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses . They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases . FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF . Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix . Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now . Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide . The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family. Protein Sci, 1998 Jun, 7(6), 1441 - 50 Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: evidence that cysteine-138 functions to initiate dithiol-disulfide interchange and to accept the reducing equivalent from reduced flavin; Veine DM et al.; Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining . The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein . Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S . It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species . C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild-type enzyme was used in this study . Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S . The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides . The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive . The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S . The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile . These results suggest that Cys138 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin. J Anim Sci, 1998 Jun, 76(6), 1651 - 61 Functional activity of antibodies at the bovine beta2-adrenoceptor; Hill RA et al.; Antibodies that can activate beta2-adrenoceptors (beta2-AR) have the potential to mimic the anabolic effects of beta-agonist drugs, such as clenbuterol . In this study, antibodies were raised in rabbits against two peptide analogues of the human beta2-adrenoceptor (beta2-AR): One peptide corresponded to the complete second outer loop of the receptor (24 amino acids; H24T), and the second peptide was a truncated version of the first (13 amino acids; H13C) . Following affinity purification, the antibodies were screened to detect interaction with beta2-AR in vitro . Membrane proteins from transformed Escherichia coli that express the beta2-AR were separated using SDS PAGE and transferred to nitrocellulose sheets . Immunoblotting revealed a single protein band (39,000 Da) that was recognized by the affinity-purified anti-H24T antibodies . However, the anti-H13C antibodies did not recognize any protein bands in immunoblots . In ligand binding studies, anti-H24T antibodies at a concentration of 50 nM, increased the affinity (KD) of the radiolabeled antagonist {125I}iodocyanopindolol for the bovine beta2-AR from 31.7 pM to 25.3 pM (P < .05) without changing the receptor number . Anti-H13C antibodies had no effect on ligand binding . In competitive ligand binding experiments, there was no effect of antibodies on the affinity of bovine beta2-AR for the agonist (-)-isoproterenol . However, functional activity of anti-H24T antibodies was demonstrated in an organ bath study . The presence of antibodies caused a leftward shift in the concentration-response curve for (-)-isoproterenol-induced relaxation of isolated bovine smooth muscle strips . Values for pD2 (-log EC50) were reduced in the presence of 10 nM antibody (8.62 +/- .11) compared to controls (8.30 +/- .08; P < .05) . Anti-H13C antibodies had no effect on (-)-isoproterenol-induced smooth muscle relaxation . These studies have demonstrated recognition, interaction, and functional activity of site-directed antibodies at the beta2-AR . Further studies will determine whether antibodies that potentiate activity at the beta2-AR may be evoked by the active immunization of cattle with the peptide H24T, and if so, whether this will cause the repartitioning of nutrients in a manner analogous to conventional beta2-agonists and thus provide an alternative to the use of xenobiotic compounds. Pediatr Infect Dis J, 1998 Jun, 17(6), 482 - 8 Prevalence of intestinal infections caused by diarrheagenic Escherichia coli in Bedouin infants and young children in Southern Israel; Porat N et al.; OBJECTIVE: To evaluate the prevalence of different Escherichia coli categories in symptomatic and asymptomatic infants and children residing in a Bedouin township in Southern Israel . METHODS: A total of 1613 stool samples were collected from a cohort of 234 infants and young children followed from birth up to 2 years of age . E . coli colonies from stool cultures from children during a diarrhea episode and those from nondiarrhea stools were hybridized with DNA probes specific for enteropathogenic, enteroinvasive, enterotoxigenic (ETEC), enteroaggregative, diffuse adherent and enterohemorrhagic strains . RESULTS: There were 1469 of 1613 (91%) samples positive for E . coli . The prevalence of E . coli categories was: enteroaggregative (25.9%); diffuse adherent (21.8%), ETEC (12.9%); enteropathogenic (7.3%); enterohemorrhagic (0.5%); and enteroinvasive (0.2%) . ETEC, expressing the heat-stable enterotoxin (ST), was the only category isolated significantly more often from cases than from controls (P = 0.005) . Of the two heat-stable enterotoxins screened in this study, only ETEC-heat stable enterotoxin (STh), the form isolated from human pathogenic ETEC, could be associated with diarrhea, whereas ETEC-STp, produced by ETEC of porcine origin, was not related to diarrhea . ETEC infections peaked during the warm, dry season . Prolonged shedding of E . coli postdiarrhea was not found in this population . CONCLUSION: The present cohort study confirmed that in this semiurban area, highly endemic for diarrheal disease, ETEC is an important cause of diarrhea in children. Protein Sci, 1998 Jun, 7(6), 1469 - 76 Guanidine hydrochloride unfolding of a transmembrane beta-strand in FepA using site-directed spin labeling; Klug CS et al.; We have used the electron spin resonance (ESR) site-directed spin-labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn-HCl) induced denaturation of several sites along a transmembrane beta-strand located in the ferric enterobactin receptor, FepA . In addition, we have continued the characterization of the beta-strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027-13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a beta-barrel motif comprised of a series of antiparallel beta-strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria . We have previously shown that a large surface loop in FepA containing the FeEnt binding site denatures independently of the beta-barrel domain (Klug CS et al., 1995, Biochemistry 34:14230-14236) . The SDSL approach allows examination of the unfolding at individual residues independent of the global unfolding of the protein . This work shows that sites along the beta-strand that are exposed to the aqueous lumen of the channel denature more rapidly and with higher cooperativity than the surface loop, while sites on the hydrophobic side of the beta-strand undergo a limited degree of noncooperative unfolding and do not fully denature even at high (e.g., 4 M) Gdn-HCl concentrations . We conclude that, in a transmembrane beta-strand, the local environment of a given residue plays a significant role in the loss of structure at each site. J R Coll Surg Edinb, 1998 Jun, 43(3), 196 - 7 An unnecessary femoral amputation? Levi N. Non-traumatic gas gangrene is extremely rare . It is commonly associated with perforation of an occult gastro-intestinal cancer . The patient's course is usually fulminant . We report a case of subcutaneous emphysema and myonecrosis of the lower extremity due to a perforated carcinoma of the large bowel . The diagnosis of colonic cancer was suspected but treatment was regrettably delayed leading to the perforation and subsequent lower extremity gas gangrene . The patient survived following a femoral amputation. Virus Genes, 1998, 16(3), 277 - 80 The gene 4 of rice yellow stunt rhabdovirus encodes the matrix protein; Luo Z et al.; The complete nucleotide sequence of the gene 4 of rice yellow stunt rhabdovirus (RYSV) was determined from cDNAs corresponding to the viral genomic RNA . Gene 4 is 913 nucleotides (nt) long, comprising a 17-nt untranslated 5' region, a 786-nt open reading frame encoding a polypeptide with a molecular mass of 29,125 Da, and a 110-nt untranslated 3' region . Western blot analysis of the RYSV proteins using the antiserum raised against the protein expressed from the cloned gene in Escherichia coli indicates that gene 4 encodes the M protein of RYSV . Comparisons of the deduced amino acid sequence of the M protein of RYSV with those of other rhabdoviruses revealed no significant homologies . However, it shared a similar basic property and a similar distribution of charges with the other rhabdovirus matrix proteins and showed a relatively closer relationship to the sonchus yellow net virus (SYNV) M1 protein. J Mol Biol, 1998 Jul 10, 280(2), 287 - 96 Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase . I . Introduction of a six-residue ion-pair network in the hinge region; Lebbink JH et al.; Comparison of the recently determined three-dimensional structures of several glutamate dehydrogenases allowed for the identification of a five-residue ion-pair network in the hinge region of Pyrococcus furiosus glutamate dehydrogenase (melting temperature 113 degrees C), that is not present in the homologous glutamate dehydrogenase from Thermotoga maritima (melting temperature 93 degrees C) . In order to study the role of this ion-pair network, we introduced it into the T . maritima enzyme using a site-directed mutagenesis approach . The resulting T . maritima glutamate dehydrogenases N97D, G376 K and N97D/G376 K as well as the wild-type enzyme were overproduced in Escherichia coli and subsequently purified . Elucidation of the three-dimensional structure of the double mutant N97D/G376 K at 3.0 A, showed that the designed ion-pair interactions were indeed formed . Moreover, because of interactions with an additional charged residue, a six-residue network is present in this double mutant . Melting temperatures of the mutant enzymes N97D, G376 K and N97D/G376 K, as determined by differential scanning calorimetry, did not differ significantly from that of the wild-type enzyme . Identical transition midpoints in guanidinium chloride-induced denaturation experiments were found for the wild-type and all mutant enzymes . Thermal inactivation at 85 degrees C occured more than twofold faster for all mutant enzymes than for the wild-type glutamate dehydrogenase . At temperatures of 65 degrees C and higher, the wild-type and the three mutant enzymes showed identical specific activities . However, at 58 degrees C the specific activity of N97D/G376 K and G376 K was found to be significantly higher than that of the wild-type and N97D enzymes . These results suggest that the engineered ion-pair interactions in the hinge region do not affect the stability towards temperature or guanidinium chloride-induced denaturation but rather affect the specific activity of the enzyme and the temperature at which it functions optimally . J Mol Biol, 1998 Jul 10, 280(2), 245 - 58 Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module; Litvinovich SV et al.; The ninth type III module of murine fibronectin was expressed in E . coli and folded into a compact homogeneous monomer whose unfolding and refolding were then investigated by fluorescence, circular dichroism, calorimetry and electron microscopy . The isolated module is unusually labile under physiological conditions . When heated at 1 deg . C/minute it exhibits an irreversible endothermic transition between 35 and 42 degrees C depending on the protein concentration . The transition is accompanied by changes in secondary and tertiary structure with partial exposure of the single tryptophan and increased binding of the hydrophobic probe, 1,8-anilinonaphthalene-sulfonate . The partially unfolded intermediate undergoes rapid self-association leading to the formation of large stable multimers that, like the original monomer, contain substantial amounts of beta sheet structure . The multimers melt and dissociate reversibly in a second endothermic transition between 60 and 90 degrees C also depending on the protein concentration . This second transition destroys the remaining secondary structure and further exposes the tryptophan . Visualization of negatively stained specimens in the electron microscope reveals that partially unfolded rmIII-9 slowly forms amyloid-like fibrils of approximately 10 nm width and indeterminate length . A subdomain swapping mechanism is proposed in which beta strands from one partially unfolded molecule interact with complementary regions of another to form oligomers and polymers . The possibility that similar interactions could play a role in the formation of fibrils by fibronectin in vivo is discussed . J Mol Biol, 1998 Jul 10, 280(2), 193 - 9 The mitochondrial processing peptidase behaves as a zinc-metallopeptidase; Luciano P et al.; The yeast mitochondrial processing peptidase (MPP) and its subunits were purified in Escherichia coli under conditions for which the enzyme retains most of its processing activity in the absence of externally added divalent cation . The holoenzyme exhibited a Km value of 1.35 microM and a Vmax value of 0.25 microM/min and was inhibited by metal chelators in a time-dependent manner . Measurement of the metal content showed that both, MPP and beta-MPP, contained 0.86 and 1.05 atoms of Zn2+ per molecule, respectively . An enzymatically inactive MPP mutant carrying a mutation of the first histidine of the putative metal-ion binding HXXEH motif in beta-MPP retained less than 0.2 atom of Zn2+ per molecule . A metal-free enzyme (apoenzyme) was prepared from the holoenzyme and shown to be devoid of any processing activity . Incubation of the apoenzyme with 50 nM and 500 nM Zn2+ restored 50% and 80% of the processing activity, respectively . However, no reactivation occurred at concentrations of Zn2+ higher than 1 microM . Addition of 500 nM Mn2+ or higher concentrations (up to 50 microM) reactivated only 50% of the processing activity . The holoenzyme was competitively inhibited by molar excess of Zn2+ (Ki of 3.1 microM) but not by molar excess of Mn2+ . Taken together, our data suggest that the authentic MPP is a Zn2+ rather than a Mn2+ metallopeptidase. J Mol Biol, 1998 Jul 10, 280(2), 185 - 92 Crystal structure of a dominant B-cell epitope from the preS2 region of hepatitis B virus in the form of an inserted peptide segment in maltodextrin-binding protein; Saul FA et al.; We report here the crystal structure of MalE-B363, a recombinant construction of maltodextrin-binding protein bearing a dominant B-cell epitope sequence from the preS2 region of the hepatitis B surface antigen . The inserted peptide sequence, which replaces the seven carboxy-terminal residues of maltodextrin-binding protein, carries the 14 amino acid residue epitope contained between residues 132 and 145 from the preS2 region . The epitope sequence is flanked on either side by additional residues that result from the genetically engineered insertion, bringing the total length of the foreign peptide to 26 amino acid residues . The hybrid protein has been previously shown to be recognised by monoclonal antibodies elicited by the native viral antigen . Three independent molecules of MalE-B363 are present in the asymmetric unit of the crystal . All 14 epitope residues could be traced for one molecule, ten epitope residues had significant electron density for the second, but no density was visible for the epitope of the third . The conformation of the amino-terminal segment of the epitope from Gln132(e) to Gly138(e) is similar in the two molecules of MalE-B363 for which the foreign peptide could be traced . Moreover, the conformation of a smaller segment, comprising residues Asp133(e) to Arg137(e), is similar to that present in the previously determined crystal structure of MalE-B133, another insertion/deletion mutant of maltodextrin-binding protein bearing the preS2 epitope . The presence of a common structural motif for the same sequence in disparate molecular environments suggests that this conformation might be present also in the native viral antigen . This could provide a structural basis to explain the cross-reactivity of anti-preS2 monoclonal antibodies with these hybrid proteins. J Invest Surg, 1997 Nov-Dec, 10(6), 349 - 55 Depression of tumor necrosis factor-alpha, interleukin-6, and interleukin-10 production: a reaction to the initial systemic hyperactivation in septic shock; Haupt W et al.; Sepsis remains a major cause of mortality in surgical intensive care units . Patients who survive the initial shock phase but die weeks later from multiple organ dysfunction still are a challenge to basic and clinical research . We addressed whether fulminant sepsis results in rapid changes (24 h) in the cellular capacity to produce cytokines in whole blood of septic patients on further stimulation after the initial systemic inflammatory response . Interleukin (IL)-6 plasma concentrations from 279 pg/mL to 5979 pg/mL confirmed the presence of a systemic inflammatory response . Anti-inflammatory IL-10 concentrations up to 275 pg/mL were detected, but there was no biologically active tumor necrosis factor-alpha (TNFalpha) detectable (by bioassay) at the time of investigation . On stimulation with Escherichia coli ex vivo, pro-inflammatory TNFalpha (130 pg/mL), IL-6 (4061 pg/mL), and anti-inflammatory IL-10 (711 pg/mL) production were markedly depressed in all patients compared with controls (2339 pg/mL, 50,319 pg/mL, and 9654 pg/mL, respectively) . Septic shock resulted in early depression of the capacity for pro- and anti-inflammatory cytokine production . Monitoring of this effect, including its relationship to outcome, may offer a target variable for therapeutic efforts to maintain or restore adequate immune reactions to improve survival. Microbiol Immunol, 1998, 42(5), 387 - 91 Serological differentiation between HCV subtypes 1a and 1b by a recombinant immunoblot assay; Schroter M et al.; Until now, no serological assay has been available for the differentiation of HCV subtypes . Since there is evidence that the subtypes differently influence the clinical course of HCV infection and the outcome of interferon therapy, we established a strip immunoblot assay (NS-4 IBA) with recombinant HCV proteins of the nonstructural 4 (NS-4) region propagated in Escherichia coli . Using this NS-4 IBA, we were able to distinguish HCV subtypes 1a and 1b, which are the most prevalent subtypes in Europe and the U.S.A . The results of the serotyping assay were compared with those obtained by nucleotide sequencing from the NS-5 region . Concordant results were observed to match 94.9% (n=100) by the NS-4 IBA and nucleotide sequencing . Discrepant results were obtained in only 5.1% (n=6) . These data indicate that HCV subtypes can be serologically distinguished, providing the possibility for easier identification of infection with different HCV subtypes. Environ Mol Mutagen, 1998, 31(4), 333 - 9 Monofunctional adenine N-3 adducts of melphalan: occurrence at a mutational hotspot sequence and resistance to removal by AlkA protein; Charles K et al.; Previous work showed that a CTAAA sequence in the supF gene of the shuttle plasmid pZ189 was a hotspot for mutagenesis by the aromatic nitrogen mustards melphalan and chlorambucil, and indirect evidence suggested adenine N-3 adducts as premutagenic lesions . In order to characterize the adducts formed at this sequence more directly, a substrate was prepared in which the three adjacent adenines in the CTAAA sequence were 3H-labeled . Following treatment of this substrate with {14C}melphalan, thermolabile adducts were depurinated and analyzed by HPLC . Only a single peak bearing both 3H and 14C label was detected and it coeluted with the single major adduct formed by the reaction of melphalan with free adenine base . Various spectrometric analyses of this species were all consistent with its identification as a monofunctional adenine N-3 adduct of melphalan . There was no evidence for any bifunctional adducts involving the labeled adenines . There was little if any release of the adenine N-3 adduct of melphalan by Escherichia coli AlkA protein, under conditions where 3-methyladenine was quantitatively released . The results support the proposal that monofunctional adenine N-3 adducts are intermediates in the generation of A.T-->T.A and A.T-->C.G transversions by aromatic nitrogen mustards. FEBS Lett, 1998 May 29, 428(3), 255 - 8 The functional properties of DsbG, a thiol-disulfide oxidoreductase from the periplasm of Escherichia coli; van Straaten M et al.; Genetic studies have recently identified DsbG, a new member of the dsb group of redox proteins, which catalyze protein disulfide bond formation in the periplasm of Escherichia coli . We now demonstrate that DsbG functions primarily as an oxidant during protein disulfide bond formation, which is consistent with the low stability of its active site disulfide bond . There are indications, however, that the substrate range of DsbG may be narrower than the other periplasmic oxidative enzymes, DsbA and DsbC . Our observations further elaborate the pathway of disulfide bond formation in E . coli. Eur J Biochem, 1998 Apr 15, 253(2), 413 - 20 Effect of single mutations on the structural dynamics of a DNA repair enzyme, the Escherichia coli formamidopyrimidine-DNA glycosylase--a fluorescence study using tryptophan residues as reporter groups; Kuznetsov SV et al.; The effects on the structure dynamics of the Escherichia coli wild-type formamidopyrimidine-DNA glycosylase (Fpg) protein of the single mutations Lys57-->Gly (FpgK57G), Pro2-->Gly (FpgP2G) and Pro2-->Glu (FpgP2E) were studied by fluorescence techniques, namely: lifetime measurements and acrylamide quenching of the fluorescence of Trp residues . The fluorescence decays of Fpg and its mutant forms were analysed by the maximum-entropy method and lifetime distributions in the range 200 ps to 9 ns were obtained . The lifetime distribution profiles of FpgK57G, FpgP2G and FpgP2E are different from that of wild-type Fpg . Both dynamic and static quenching by acrylamide were observed for all the proteins . At 20 degrees C, the bimolecular collisional quenching rate constant of the FpgP2E fluorescence by acrylamide was only 0.8 M(-1) s(-1) as compared to about 1.4 M(-1) s(-1) for the three other proteins . At 6 degrees C, all the spectroscopic properties of these four proteins are about the same . The analysis of experimental data demonstrates that all three mutations induce a structural reorganization of the Fpg protein . However, only the P2E mutation lead to a reduced accessibility of some Trp residues to acrylamide quenching . It is concluded that the single P2E replacement induces a conformational change leading to a more rigid globular structure as opposed to the wild type and K57G and P2G mutations . The influence of the single mutations on the enzyme activities of the Fpg protein is discussed. Eur J Biochem, 1998 Apr 15, 253(2), 371 - 81 Monitoring of RNA polymerase-DNA UP element interaction by a fluorescent probe conjugated to alpha subunit; Ozoline ON et al.; The carboxy-terminal domain (CTD) of Escherichia coli RNA polymerase alpha subunit was specifically modified by a reporter label, fluorescein mercuric acetate (FMMA), conjugated to Cys269 on the surface of UP element recognition helix . The modified enzyme was used to investigate RNA polymerase interaction with different promoters, either with or without an UP element . In a single-round transcription assay, the activity of modified RNA polymerase was found to decrease as measured with rrnBP1, trpP and lacP2 promoters but not with many other promoters including mutant rrnBP1 without the UP element, supporting the idea that Cys269 or the domain including Cys269 is involved in UP element recognition . Both trpP and lacP2 have sequence similarity to the rrnBP1 UP element . The chemical modification of RNA polymerase, however, did not affect an apparent equilibrium dissociation constant with rrnBP1, as measured by gel-retardation assays, indicating that the DNA-binding ability is retained even after FMMA conjugation . Interaction with the rrnBP1 UP element led to substantial alterations in the spectral parameters of the reporter label, which are different from those induced by complex formation with promoters without UP elements . A pronounced spectral blue shift suggests that the labeled surface of alphaCTD closely approaches the charged UP DNA helix . These observations imply that the fluorescent labeling at Cys269 can be used as a good tool for monitoring the presence or absence of an UP element in a given promoter . Spectral parameters of the label displayed the spectral blue shift when the modified RNA polymerase interacted with trpP, supporting the prediction that this promoter carries an rrnBP1-type UP element. Eur J Biochem, 1998 May 1, 253(3), 698 - 705 Methanol:coenzyme M methyltransferase from Methanosarcina barkeri--identification of the active-site histidine in the corrinoid-harboring subunit MtaC by site-directed mutagenesis; Sauer K et al.; The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group . The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously . We report here on the overproduction of the apoprotein of MtaC in E . coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methyl-cob(III)alamin, and on the properties of the reconstituted corrinoid protein . Reconstituted MtaC was found to contain 1 mol bound cobamide/mol . EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+ of the bound corrinoid . This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues . The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA . In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M . barkeri . M . barkeri contains an isoenzyme of MtaA designated MtbA . The isoenzyme reacted with MtaC with only 2.5% of the activity of MtaA. Eur J Biochem, 1998 May 1, 253(3), 653 - 8 Pigment-binding properties of the recombinant photosystem II subunit CP26 reconstituted in vitro; Ros F et al.; CP26 is the most recently described antenna protein in higher plants which has been reported to be involved in xanthophyll-dependent regulation of the light-harvesting function but is largely unknown due to the difficulties of purification . In this study we have overexpressed in Escherichia coli the Lhcb5 gene product and reconstituted CP26 in vitro by refolding the recombinant protein in the presence of chlorophyll a, chlorophyll b and xanthophylls . The resulting pigment-protein complex is stable enough to be isolated by partially denaturing gel electrophoresis . Reconstitution and isolation conditions for CP26 are similar to those used for other chlorophyll a/b complexes like the major light-harvesting complex of photosystem II (LHCII) and CP29; however, CP26 differs with regard to its lower specificity in carotenoid binding . Most significantly, rather stable recombinant CP26 can be reconstituted containing violaxanthin as the only carotenoid . This enhanced plasticity with respect to carotenoid binding is consistent with CP26 being the major binding protein of violaxanthin involved in the xanthophyll cycle . The availability of recombinant CP26 opens the way to a better characterisation of this pigment-protein complex with regard to its biochemistry and its physiological functions. Eur J Pharmacol, 1998 Apr 24, 347(2-3), 319 - 27 Kinetics of alkylation of cloned rat alpha1-adrenoceptor subtypes by chloroethylclonidine; Xiao L et al.; We quantified and compared the rates at which chloroethylclonidine (CEC) inactivated cloned rat alpha1A, alpha1B-, and alpha1D-adrenoceptors . Membranes from cells transfected with one of the three cloned alpha1-adrenoceptors were incubated for various intervals with 100 microM chloroethylclonidine at 10 degrees C, 25 degrees C or 37 degrees C . The fraction of receptors alkylated by chloroethylclonidine was determined by {3H}prazosin binding . Chloroethylclonidine fully inactivated each alpha1-adrenoceptor subtype via a first order reaction . Alkylation by chloroethylclonidine was markedly slower for the alpha1A-adrenoceptor vs . the other two subtypes (rate constants in 10(-3) min(-1) at 10 degrees C: 0.99 +/- 0.01 (alpha1A), 7.26 +/- 0.15 (alpha1B), and 7.01 +/- 0.12 (alpha1D)) . Despite differences in rate, activation energies for alkylation were similar among subtypes . suggesting a similar binding sites for chloroethylclonidine . Computer simulations of kinetic data in mixed receptor populations and experiments with membranes from rat brain showed that nonlinear curve fitting could distinguish relative proportions of alpha1A-adrenoceptor vs . the other two subtypes . We conclude that measurement of the rate of alkylation by chloroethylclonidine, rather than the total amount of alkylation, is most useful in distinguishing the relative proportion of alpha1A-adrenoceptor in tissues. Rom J Physiol, 1997 Jan-Dec, 34(1-4), 95 - 101 Effects of monosodium glutamate on blood neutrophils phagocytic activity and phagocytic response in mice; Hriscu M et al.; Previous researches of our laboratories (1945, 1946, 1947) have shown that direct electrical stimulation of the tubero-mammillary hypothalamic area in dogs enhances the blood neutrophils phagocytic activity and the phagocytosis exhibiting leukocytes percent . After electrolytic damage of the same area, phagocytic activity decreases and phagocytic response is suppressed (1985, 1988) . In the present work, we performed in mice extensive chemical lesions of the arcuate nucleus, by means of the neonatal treatment with monosodium glutamate (MSG) . The experiment was carried out on 23 new-born mice . 15 mice were injected with MSG (G group), the other 8, serving as control group, received isotonic saline solution (C group) . The studied parameters were, in both groups, the weight evolution of the animals, the blood neutrophils and lymphocytes percentual variation and the neutrophils' phagocytic activity, tested in vitro, expressed through the number of bacteria engulfed by 100 neutrophils and through the phagocyting neutrophils percentage . Phagocytic activity was tested in whole heparinised blood, against E . coli . Phagocytic response was elicited by i.p . injecting 0.05 ml bacterial suspension and was tested four hours later . The results show that the arcuate nucleus has little influence upon maintaining basal phagocytic activity--that does not significantly decrease after its chemical damage--, but plays a decisive role in triggering the phagocytic response . The neonatal MSG treatment also determines a decrease of the blood lymphocytes percentage and induces obesity in up to 30 days old mice pups. Pharm Dev Technol, 1998 May, 3(2), 269 - 76 Stabilization of dichloromethane-induced protein denaturation during microencapsulation; Raghuvanshi RS et al.; This paper describes the denaturation of protein drugs by dichloromethane (DCM) during the primary emulsification step of the microencapsulation process using biodegradable polymer matrix for controlled-release application . It was found that interaction of proteins such as tetanus toxoid (TT), diphtheria toxoid (DT), ovine growth hormone (oGH), and human chorionic gonadotropin-based antifertility vaccine (beta-hCG-TT) with DCM during primary emulsification stages of particle formulation led to the precipitation of the proteins at the aqueous organic interface with concomitant reduction in their immunoreactivity . On the other hand, the B subunit of E . coli enterotoxin (LTB) was found to be comparatively stable toward the denaturing action of DCM . Attempts were made to overcome the DCM-induced denaturation by incorporation of stabilizers during the primary emulsification step of the particle formulation . Of the many additives tested to overcome the DCM-induced denaturation of proteins, serum albumins and polyvinyl alcohol (PVA) showed promising results in terms of retention of the immunoreactivity of the protein . TT stabilized by the incorporation of serum albumin during the primary emulsification step not only showed immunoreactivity in vitro, but also invoked antibody titers in rats comparable to those obtained for the native protein molecules . Incorporation of 2.5% of serum albumins in the internal aqueous phase not only protected the protein from the degradative action of DCM but also led to stabilized primary emulsion, which is necessary for uniform entrapment of protein drugs in the polymer matrix. Novartis Found Symp, 1998, 213, 142 - 55; discussion 155-9 The nested networks of brains and minds; Barlow H; The reductionist approach to the brain shows promise of revolutionizing our ideas about what single neurons can do . A spine on a cortical pyramidal cell is about the size of a single Escherichia coli, and if the internal machinery of a spine is anything like as well organized as that of E . coli, the whole pyramidal cell with its 5000 spines must be capable of computations an order of magnitude more complex than those demanded of the neurons used for current models of the brain . These computations might enable single neurons to detect spatiotemporal patterns, i.e . Hebb's 'phase sequences' . Reductionism is apparently limited because its drive is to look for explanations at lower levels in the organizational tree . For this purpose it often uses isolated preparations in which such lower levels can be studied but higher levels cannot, because they have been thrown down the sink . Reductionism will never lead us to understand organization and interaction in parts discarded or ignored, and this must include the interactions between individual human minds that are crucial for understanding human society . Our brains possess a 'commentary system', a mechanism that can make reports on the internal status of some parts of the brain . This makes possible networks of minds, and the present meeting is such a network whose interactions are being recorded for posterity . On a grander scale such networking creates a cultural forum where communal goals and purposes are formulated, disseminated, modified, and often perpetuated in lasting form . The resulting group behaviour has obvious survival value, and is perhaps the feature that distinguishes humans most clearly from other species. Biosci Rep, 1998 Feb, 18(1), 39 - 48 Computational observation of an ion permeation through a channel protein; Suenaga A et al.; The ion permeation process, driven by a membrane potential through an outer membrane protein, OmpF porin of Escherichia coli, was simulated by molecular dynamics . A Na+ ion, initially placed in the solvent region at the outer side of the porin channel, moved along the electric field passing through the porin channel in a 1.3 nsec simulation; the permeation rate was consistent with the experimentally estimated channel activity (10(8)-10(9)/sec) . It this simulation, it was indicated that the ion permeation through the porin channel proceeds by a "push-out" mechanism, and that Asp113 is an important residue for the channel activity. Mutat Res, 1998 Jun, 407(3), 253 - 9 Hydrogen peroxide induces protection against lethal effects of cumene hydroperoxide in Escherichia coli cells: an Ahp dependent and OxyR independent system? Asad NR, Asad LM, Silva AB, Felzenszwalb I, Leitao AC. Pretreatment with 2.5 mM H2O2 protects bacterial cells against cumene hydroperoxide killing . This response is independent of the OxyR system, but possibly involves the participation of Ahp protein, since ahp mutants are not protected . Treatment of bacterial cells with high H2O2 concentrations caused an alteration on the electrophoretic profile of the smaller subunit (22-kDa) of Ahp . This alteration does not require novel gene products and is not dependent on the OxyR protein . In this way, we propose that the modification of the 22-kDa subunit of Ahp by high H2O2 concentration may be responsible for the protection against the lethal effects of cumene hydroperoxide. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 8016 - 9 Characterizing semilocal motions in proteins by NMR relaxation studies; Fischer MW et al.; The understanding of protein function is incomplete without the study of protein dynamics . NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies . The use of 15N relaxation to study backbone dynamics has become virtually standard . Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions . This allows the nature of local motions to be characterized in proteins . As an example, semilocal rotational motion was detected for part of a helix of the protein Escherichia coli flavodoxin. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7969 - 74 The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA; Connelly JC et al.; Hairpin structures can inhibit DNA replication and are intermediates in certain recombination reactions . We have shown that the purified SbcCD protein of Escherichia coli cleaves a DNA hairpin . This cleavage does not require the presence of a free (3' or 5') DNA end and generates products with 3'-hydroxyl and 5'-phosphate termini . Electron microscopy of SbcCD has revealed the "head-rod-tail" structure predicted for the SMC (structural maintenance of chromosomes) family of proteins, of which SbcC is a member . This work provides evidence consistent with the proposal that SbcCD cleaves hairpin structures that halt the progress of the replication fork, allowing homologous recombination to restore DNA replication. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7963 - 8 Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase; Bringaud F et al.; Trypanosomatids are parasitic protists that have an ATP-dependent glycolysis with no indication of PPi-dependent metabolism . Most of the glycolysis takes place in peroxisome-like organelles, the glycosomes . We characterized in Trypanosoma brucei a single-copy gene encoding a PPi-dependent enzyme, pyruvate, phosphate dikinase (PPDK), which was expressed functionally in Escherichia coli . Specific antibodies detected a 100-kDa protein in procyclic forms but not in mammalian forms of T . brucei, indicating a differential expression . Glycosomal localization of PPDK was determined by immunofluorescence analysis and was confirmed by Western blot analysis on glycosomal fractions by using anti-PPDK antibodies . Expression and localization of recombinant PPDKs in procyclic forms of T . brucei showed that the AKL motif at the C-terminal extremity of PPDK is necessary for glycosomal targeting . PPDK was detected in every trypanosomatid tested-Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Phytomonas, Crithidia and Leishmania-with a good correlation between amount of protein and enzymatic activity . The precise role of PPDK in trypanosomatid carbohydrate metabolism remains to be clarified. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7876 - 81 Structural similarities between topoisomerases that cleave one or both DNA strands; Berger JM et al.; Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex . Both types have been proposed to use an "enzyme-bridging" mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment . Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA. J Mol Biol, 1998 Jul 3, 280(1), 103 - 16 Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein; Malhotra A et al.; Cryo-electron microscopy of the ribosome in different binding states with mRNA and tRNA helps unravel the different steps of protein synthesis . Using over 29,000 projections of a ribosome complex in single-particle form, a three-dimensional map of the Escherichia coli 70 S ribosome was obtained in which a single site, the P site, is occupied by fMet-tRNAfMet as directed by an AUG codon containing mRNA . The superior resolution of this three-dimensional map, 14.9 A, has made it possible to fit the tRNA X-ray crystal structure directly and unambiguously into the electron density, thus determining the locations of anticodon-codon interaction and peptidyltransferase center of the ribosome . Furthermore, at this resolution, one of the distinctly visible domains corresponding to a ribosomal protein, L1, closely matches with its X-ray structure . Scand J Immunol, 1998 Jun, 47(6), 609 - 14 Placental transfer of IgG and IgG subclass antibodies anti-purified Escherichia coli LPS O16, O6 and O111; Nagao AT et al.; We evaluated 22 paired maternal and cord sera regarding the presence of IgG and IgG subclasses against purified Escherichia coli LPS O6, O16 and O111 employing ELISA for titre and avidity analysis, isoelectric focusing associated with affinity-blotting for spectrotypic analysis, and the Western-blotting technique for recognition of the various bands in lipopolysaccharide (LPS) . Levels of anti-LPS IgG antibodies in cord sera were equivalent to their respective maternal sera, showing a significant correlation (P < 0.0001) . IgG1 antibody levels were higher in cord sera than in maternal sera (P < 0.005 for anti-O111, P < 0.05 for anti-O16 and P < 0.02 for anti-O6) . Cord IgG2 antibody levels were not different from the maternal levels (P > 0.1) . The levels of IgG3 and IgG4 were undetectable . The avidity of anti-O6 and anti-O111 IgG in 10 cord sera showed an extremely significant correlation with maternal antibody avidity (P < 0.0001) . Identical patterns of recognition were found in the paired samples analysed by Western blotting . Most of the serum samples recognized the O-repetitive chains and also the region corresponding to core and lipid A . Although the antibody spectrotypes varied among individuals, paired cord and maternal serum samples showed identical patterns . Our findings suggest the occurrence of placental transfer of IgG antibodies against LPS O6, O16 and O111, mainly involving the IgG1 or IgG2 subclasses. J Infect Dis, 1998 Jul, 178(1), 185 - 90 Enteroaggregative Escherichia coli as a potential cause of diarrheal disease in adults infected with human immunodeficiency virus; Wanke CA et al.; Stools of 68 human immunodeficiency virus (HIV)-infected adults with diarrhea and 60 without diarrhea were examined for enteroaggregative Escherichia coli (EAggEc) by HeLa cell adherence assay . EAggEc were present in stools of 30 patients with and 18 without diarrhea (P = .05) . CD4 cell counts of patients with EAggEc and diarrhea were significantly lower than those of patients with EAggEc without diarrhea (P = .02) . There was no difference in the mean duration of diarrheal symptoms or in the number of stools per day between patients with EAggEc and those without . None of the EAggEc strains were positive by polymerase chain reaction for adherence fimbria, but 11 strains were positive for EAggEc heat-stable toxin EAST/1 . Of the EAggEc strains, 51% were resistant to trimethoprim-sulfamethoxazole and 65% were resistant to ampicillin . EAggEc may be a pathogen in HIV-infected patients with diarrhea; HIV-infected patients with EAggEc appear to be more symptomatic when HIV disease is more advanced. J Infect Dis, 1998 Jul, 178(1), 178 - 84 Induction of apoptosis in normal human renal tubular epithelial cells by Escherichia coli Shiga toxins 1 and 2; Kiyokawa N et al.; The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated . HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs . Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs . Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx . DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process . These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E . coli infection. J Infect Dis, 1998 Jul, 178(1), 127 - 37 Expression of chemokines and induction of rapid cell death in human blood neutrophils by Mycobacterium tuberculosis; Kasahara K et al.; To elucidate the role of neutrophils in the early inflammatory response to mycobacterial infection, expression of chemokines interleukin (IL)-8 and macrophage inflammatory protein-1alpha (MIP-1alpha) was examined in human blood neutrophils in response to the lipopolysaccharide (LPS) of Escherichia coli, which induces acute inflammation, or to Mycobacterium tuberculosis or purified protein derivative (PPD), which induce chronic mycobacterial inflammation . Neutrophils stimulated with LPS, M . tuberculosis, or PPD expressed both IL-8 and MIP-1alpha . Expression of IL-8 and MIP-1alpha was lower after stimulation with M . tuberculosis or PPD than after stimulation with LPS, but the kinetics of expression did not differ significantly . In contrast, both M . tuberculosis and PPD with tumor necrosis factor-alpha induced neutrophils to undergo rapid cell death, which might remove neutrophils and activate macrophages at sites of mycobacterial inflammation . The findings suggest that neutrophils play important roles in the host defense against mycobacterial infection. Eur J Biochem, 1998 May 15, 254(1), 168 - 71 Structural determination of the O-antigenic polysaccharide from Escherichia coli O141; Farnback M et al.; The structure of the O-antigenic polysaccharide from Escherichia coli O141 has been determined . NMR spectroscopy and sugar and methylation analyses were the principal methods used . The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple-bond connectivity (HMBC-) NMR experiments . The polysaccharide is composed of pentasaccharide repeating units with 1 O-acetyl group/repeating unit . The following structure, where Rha is 6-deoxymannose is concluded: carbohydrate sequence {see text}. Eur J Biochem, 1998 May 15, 254(1), 154 - 9 Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin); Ziegler K et al.; Cyanophycin (multi-L-arginyl-poly-L-aspartate), a water-insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis . The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described . In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa . The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size-exclusion chromatography, suggesting that the active form is a homodimer . During catalysis, ATP is converted to ADP . The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp . PCC 6803 . The C-terminal 60% of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl-poly(gamma-glutamate) . Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin-like material . The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin. Eur J Pharmacol, 1998 Apr 10, 346(2-3), 283 - 90 Tetracycline inhibits the nitric oxide synthase activity induced by endotoxin in cultured murine macrophages; D'Agostino P et al.; Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro . The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation . For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM . The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-{4,5-dimethylthiazol-2yl}-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot . However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events . The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription . The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds. Eur J Pharmacol, 1998 May 8, 348(2-3), 247 - 56 Nitric oxide enhances prostaglandin production in ethanol-induced gastric mucosal injury in rats; Franco L et al.; The interaction between endogenous nitric oxide (NO), elicited by administration of Escherichia coli lipopolysaccharide, and cyclooxygenase system, in ethanol-induced injury in rat gastric mucosa, was investigated . Administration of graded doses of lipopolysaccharide reduced the gastric mucosal injury in response to ethanol . The ex vivo production of both nitrite and prostaglandin E2 was increased in dose-related manner by lipopolysaccharide . Pretreatment with dexamethasone, L-N6-(1-Iminoethyl)lysine(dihydrochloride) and L-NG-nitro arginine methyl ester inhibited the protection associated with lipopolysaccharide treatment and the ex vivo production of both, nitrite and prostaglandin E2 . The pretreatment with L-arginine counteracted the decrease of nitrite and prostaglandin E2 production in lipopolysaccharide-treated rats in which nitric oxide synthesis was blocked by L-N6-(1-Iminoethyl)lysine(dihydrochloride) . Administration of sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine caused a dose related enhancement in the accumulation of prostaglandin E2 . Indomethacin administration and N-(2-Cyclohexyloxy-4-nitrophenyl)methanesulfonamide were ineffective in suppressing lipopolysaccharide-mediated protection against ethanol-induced damage, and in suppressing ex vivo increase of nitrite whereas the ex vivo increase of prostaglandin E2 was prevented in a dose-related fashion . These results indicate that in ethanol-induced rat gastric injury, endogenous NO elicited by lipopolysaccharide or released by NO donors is able to activate the cyclooxygenase pathway, and the protective effect of lipopolysaccharide is dependent upon NO formation. Prog Biophys Mol Biol, 1997, 68(2-3), 121 - 44 Structure and function of molybdopterin containing enzymes; Romao MJ et al.; Molybdopterin containing enzymes are present in a wide range of living systems and have been known for several decades . However, only in the past two years have the first crystal structures been reported for this type of enzyme . This has represented a major breakthrough in this field . The enzymes share common structural features, but reveal different polypeptide folding topologies . In this review we give an account of the related spectroscopic information and the crystallographic results, with emphasis on structure-function studies. Vet Q, 1998, 20 Suppl 2, S20 - 4 Diagnostic potential of Mab-based ELISAs for antibodies to non-structural proteins of foot-and-mouth disease virus to differentiate infection from vaccination; Brocchi E et al.; This paper summarises the development of monoclonal antibody (Mab)-based immunoassays measuring antibodies to non-structural proteins of FMDV to differentiate infection from vaccination . Of the three non-structural proteins 2C, 3C and 3ABC evaluated in this study, the polypeptide 3ABC was the most immunogenic . Three ELISAs for the detection of antibodies to 3ABC were developed . Two assays rely on the competition of test sera against either a anti-3A Mab or against antisera to 3ABC raised in rabbits and guinea-pigs . The third, 3ABC Mat-ELISA, based on the direct binding of antibodies to the 3ABC trapped by a specific Mab, provided the best combination of specificity and sensitivity . The 3ABC Mat-ELISA was extensively validated for cattle, either in experimental and in field conditions, showing specificity of 99% in vaccinated and in naive cattle and the capacity to detect silent infections in FMD-vaccinated populations . The test showed similar specificity and sensitivity in experimentally vaccinated and infected sheep. Vet Q, 1998, 20 Suppl 2, S9 - 11 Antibody to the nonstructural proteins of foot-and-mouth disease virus in vaccinated animals exposed to infection; Mackay DK et al.; Cattle which have been infected with foot-and-mouth disease (FMD) virus can be differentiated from those that have been vaccinated on the basis of the detection of antibody to one or more of the non-structural (NS) proteins of the virus . Cattle which have been protected by vaccination can become persistently infected with FMD virus (FMDV) without ever showing clinical signs . Vaccinated, protected cattle which are persistently infected cannot be distinguished from animals that merely have been vaccinated on the basis of serological tests for antibody to the structural proteins of FMDV . Sera were collected from groups of cattle for varying periods after exposure to infection under experimental conditions . On the basis of isolation of virus from probang samples collected during the course of the experiments it was possible to classify the cattle according to the following criteria; naive, infected and eliminated the virus (convalescent), infected and persistently infected with FMDV (carriers), vaccinated alone, vaccinated and either convalescent or carrier . Sera were examined for antibody to the NS proteins Lb, 2C, 3A, 3D, and 3ABC by an indirect profiling ELISA using E . coli-expressed fusion proteins as antigens . Considerable variation was observed in the antibody response to NS proteins of both naive and vaccinated animals following infection . The extent of individual variation was so great that convalescent animals could not be differentiated from carrier animals on the basis of their antibody response to any of the NS proteins examined . The majority of vaccinated, protected animals showed an antibody response to NS proteins, particularly 3ABC, following exposure to infection . However, the carrier state was demonstrated in some vaccinated, protected animals in which no antibody response to any of the NS proteins could be detected . The detection of antibody to NS proteins can therefore be used on a group, or herd, basis to detect circulation of virus in a vaccinated population but further investigations in the field are required to determine the sampling level necessary for statistical acceptance . On an individual animal basis, however, freedom from antibody to NS proteins in a vaccinated animal, or an animal of unknown history, does not necessarily imply that the animal is free from infection with FMD virus and, furthermore, the titre of antibody to NS proteins is not a useful predictive measure of whether or not an infected animal has successfully eliminated the virus. J Comp Pathol, 1998 May, 118(4), 337 - 45 In-vitro studies on mechanisms of immunosuppression associated with bovine respiratory syncytial virus; Keles I et al.; Bovine respiratory syncytial virus (BRSV) depressed the proliferative reactivity of normal ovine peripheral blood lymphocytes to phytohaemagglutinin (PHA) . This BRSV-induced reduction in proliferative reactivity was not reversed or ameliorated by the addition of (1) indomethacin or flunixin meglumine, substances known to inhibit the production of prostaglandins, or (2) the cytokines, interleukin-1 (IL-1) and interleukin-2 (IL-2), or (3) rat growth factor . The results suggest that the suppression of ovine lymphocyte reactivity to PHA associated with BRSV was not caused by the release of cyclooxygenase products such as prostaglandins, or the production of inhibitors of IL-1 or IL-2. Jpn J Antibiot, 1997 Nov, 50(11), 855 - 61 {Predictive factors for development of hemolytic uremic syndrome (HUS) and early intensive treatments for prevention of HUS enterohemorrhagic Escherichia coli infection}; Oshima T; Predictive factors for the development of hemolytic uremic syndrome (HUS) were evaluated in 88 inpatients who suffered from enterohemorrhagic E . coli infections in the outbreak in Sakai, 1996 . All in- and outpatients received oral or intravenous fosfomycin within acute phase of hemorrhagic colitis, and HUS complicated 1.4% of them . Persistence of bloody stools and diarrhea were longer in HUS patients than in non-HUS patients, but persistence of abdominal pain was not different in either group . Leukocytosis with leukocyte counts over 15,000/microliters and/or elevated CRP level over 2.0 mg/dl at admission, and fever and/or vomiting in the course of hemorrhagic colitis were more frequent in HUS patients than in non-HUS patients . Early intensive treatments including gammaglobulin, urinastatin, aspirin, and dipyridamole were employed in 34 high risk patients for prevention of HUS . These patients were estimated to be at risk of developing HUS because of incomplete HUS, nephropathy, elevated LDH level, thrombocytopenia, or age younger than two years old . These treatments were clinically effective. Gene, 1998 Jul 3, 214(1-2), 121 - 9 Three alternatively spliced mouse slow skeletal muscle troponin T isoforms: conserved primary structure and regulated expression during postnatal development; Jin JP et al.; We have cloned and sequenced full-length cDNAs encoding mouse slow skeletal muscle troponin T (sTnT) . Alternative mRNA splicing-generated two high Mr isoforms and one low Mr sTnT isoform differing in the NH2-terminal primary structure have been identified by Western blotting, reverse transcription-polymerase chain reaction and cDNA cloning/expression analyses . Together with a 5'-alternative exon that was also found in human sTnT encoding an 11-amino-acid acidic segment, the results revealed a novel alternative splicing pathway to include or exclude a three-base segment to generate additional sTnT isoforms with NH2-terminal charge variations . Overriding the phylogenetic divergence, primary structure of sTnT is better conserved between mammalian and avian species than that of cardiac, fast and skeletal muscle TnTs from one species . Western blots demonstrate four expression patterns of sTnT during postnatal skeletal muscle development: (1) a decrease to a non-detectable level in mouse masseter, (2) an increase to become the sole TnT in sheep masseter, (3) an increase of the total level as well as the proportion of the low Mr isoform in sheep diaphragm and, (4) no significant change in total level or high/low Mr isoform ratio in sheep gastrocnemius . The highly conserved primary structure and fiber type-specific and developmentally regulated expression of sTnT indicate a physiological importance of this under-studied member of the TnT gene family. J Biol Chem, 1998 Jul 10, 273(28), 17933 - 9 Biochemical coupling between the DrrA and DrrB proteins of the doxorubicin efflux pump of Streptomyces peucetius; Kaur P et al.; The drrAB operon of Streptomyces peucetius encodes for resistance to the antibiotics doxorubicin and daunorubicin . Subcloning of the drrAB genes in Escherichia coli has previously been shown to result in expression of DrrA and DrrB proteins and resistance to doxorubicin in a sensitive strain of E . coli . DrrA, a peripheral membrane protein, binds ATP in a UV-catalyzed reaction in a doxorubicin-dependent manner; DrrB, a hydrophobic protein, is localized to the inner membrane of E . coli (Kaur, P . (1997) J . Bacteriol . 179, 569-575) . The present study provides evidence that DrrB, the membrane component of the complex, is stably maintained in the cell only if DrrA is present . Furthermore, it was found that the catalytic component DrrA is in an active conformation only when it is in a complex with DrrB . In a subclone containing the drrB gene by itself, no DrrB protein could be detected, although a translational fusion of the first 15 amino acids of DrrB to beta-galactosidase indicated that DrrB is translated in the absence of DrrA . Upon co-transformation with a plasmid containing the drrA gene in trans, DrrB could again be detected in these cells . UV cross-linking studies with {alpha-32P}ATP showed that only the membrane-bound form of DrrA in cells containing both DrrA and DrrB was in a conformation competent to bind ATP . Chemical cross-linking studies also provided direct evidence for interaction between the two proteins . Based on these analyses, a model for interaction between DrrA and DrrB proteins is presented. J Biol Chem, 1998 Jul 10, 273(28), 17839 - 45 Characterization of recombinant CD45 cytoplasmic domain proteins . Evidence for intramolecular and intermolecular interactions; Felberg J et al.; CD45 is a transmembrane two-domain tyrosine phosphatase required for efficient signal transduction initiated by lymphocyte antigen receptors . As with most transmembrane two-domain phosphatases, the role of the second phosphatase domain is unclear . In this study, recombinant CD45 cytoplasmic domain proteins purified from bacteria were used to evaluate the function of the individual phosphatase domains . A recombinant protein expressing the membrane-proximal region, first phosphatase domain, and spacer region of CD45 (rD1) was catalytically active and found to exist primarily as a dimer . In contrast to this, a recombinant protein expressing the spacer region, the second phosphatase domain and the carboxy tail of CD45 (rD2) existed as a monomer and had no catalytic activity against any of the substrates tested . Comparison of rD1 with the recombinant protein expressing the entire cytoplasmic domain of CD45 (rD1/D2) indicated that rD1/D2 was 2-3-fold more catalytically active, was more thermostable, and existed primarily as a monomer . Limited trypsin digestion of rD1/D2 provided evidence for a noncovalent association between an N-terminal 27-kDa fragment and a C-terminal 53-kDa fragment, suggesting an intramolecular interaction . Furthermore, rD1 was found to specifically associate with rD2 in an in vitro binding assay . Taken together, these data provide evidence for an intramolecular interaction occurring in the cytoplasmic domain of CD45 . In the absence of the C-terminal region containing the second phosphatase domain, intermolecular interactions occur, resulting in dimer formation. J Biol Chem, 1998 Jul 10, 273(28), 17680 - 8 Isoenzymes of pyruvate dehydrogenase phosphatase . DNA-derived amino acid sequences, expression, and regulation; Huang B et al.; Pyruvate dehydrogenase phosphatase (PDP) is one of the few mammalian phosphatases residing within the mitochondrial matrix space . It is responsible for dephosphorylation and reactivation of the pyruvate dehydrogenase complex (PDC) and, by this means, is intimately involved in the regulation of utilization of carbohydrate fuels in mammals . PDP is a dimeric enzyme consisting of catalytic and regulatory subunits . The catalytic subunit of PDP is a Mg2+-dependent enzyme homologous to the cytosolic phosphatases of the 2C family . In the present study, we isolated two cDNAs encoding for mitochondrial phosphatases . The first cDNA is highly homologous to the previously identified cDNA encoding for the catalytic subunit of PDP (PDP1) . The second cDNA encodes a previously unknown catalytic subunit of PDP (PDP2) . The new phosphatase, expressed as the recombinant protein in Escherichia coli, shows strict substrate specificity toward PDC and does not use phosphorylated branched chain alpha-ketoacid dehydrogenase as substrate . Like PDP1, PDP2 is a Mg2+-dependent enzyme, but its sensitivity to Mg2+ ions is almost 10-fold lower than that of PDP1 . In contrast to PDP1, PDP2 is not regulated by Ca2+ ions . Instead, it is sensitive to the biological polyamine spermine, which, in turn, has no effect on the enzymatic activity of PDP1 . Western blot analysis of PDP extracted from mitochondria isolated from liver and skeletal muscle revealed that PDP1 is predominantly expressed in mitochondria from skeletal muscle, whereas PDP2 is much more abundant in the liver rather than muscle mitochondria . Both isoenzymes are expressed in mitochondria from 3T3-L1 adipocytes, but the level of expression of PDP2 is considerably higher . These observations are consistent with previous findings on the enzymatic parameters of PDP in adipose tissue . Thus, our results provide the first evidence that there are at least two isoenzymes of PDP in mammals that are different with respect to tissue distribution and kinetic parameters and, therefore, are likely to be different functionally. J Biol Chem, 1998 Jul 10, 273(28), 17671 - 9 Subunit interactions within an expressed regulatory domain of chicken skeletal myosin . Location of the NH2 terminus of the regulatory light chain by fluorescence resonance energy transfer; Saraswat LD et al.; The regulatory domain (RD), or neck region of the myosin head, consists of two classes of light chains that stabilize an alpha-helical segment of the heavy chain . RD from chicken skeletal muscle myosin was prepared in Escherichia coli by coexpression of a 9-kDa heavy chain fragment with the essential light chain . Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the complex . The affinity of RD for divalent cations was determined by measuring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium . The complex bound divalent cations with high affinity, similar to the association constants determined for native myosin . The intrinsic fluorescence of the tryptophans could be used as a donor to measure the fluorescence resonance energy transfer distance to a single labeled cysteine engineered at position 2 on RLC . Dansylated Cys2 could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an acceptor probe . These fluorescence resonance energy transfer distances (24-30 A), together with a previous measurement between Cys2 and Cys155 (Wolff-Long, V . L., Tao, T., and Lowey, S . (1995) J . Biol . Chem . 270, 31111-31118) suggest a location for the NH2 terminus of RLC that appears to preclude a direct interaction between the phosphorylatable serine and specific residues in the COOH-terminal domain. J Biol Chem, 1998 Jul 10, 273(28), 17604 - 9 Crystal structure and mutational analysis of the Escherichia coli putrescine receptor . Structural basis for substrate specificity; Vassylyev DG et al.; PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli . We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution . The PotF molecule has dimensions of 54 x 42 x 30 A and consists of two similar globular domains . The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD . Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions . The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins . The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition. J Biol Chem, 1998 Jul 10, 273(28), 17418 - 24 Biosynthesis of pteridines in Escherichia coli . Structural and mechanistic similarity of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase; Haussmann C et al.; An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate . The gene was subsequently designated folB . The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E . coli chromosome . The folX and folB gene products were both expressed to high yield in recombinant E . coli strains, and the recombinant proteins were purified to homogeneity . Both enzymes form homo-octamers . Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2' of dihydroneopterin and dihydromonapterin at appreciable velocity . Epimerase catalyzes the epimerization of carbon 2' in the triphosphates of dihydroneopterin and dihydromonapterin . However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate . Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions . We propose that the polarization of the 2'-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity . A deletion mutant obtained by targeting the folX gene of E . coli has normal growth properties on complete medium as well as on minimal medium . Thus, the physiological role of the E . coli epimerase remains unknown . The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase . However, the genome of H . influenzae does not specify a dihydroneopterin-triphosphate epimerase. J Biol Chem, 1998 Jul 10, 273(28), 17406 - 10 Truncation of amino acids 12-128 causes deregulation of the phosphatase activity of the sensor kinase KdpD of Escherichia coli; Jung K et al.; The kdpFABC operon, which encodes the structural genes for the high affinity K+ transport complex KdpFABC, is regulated by the sensor kinase KdpD and the response regulator KdpE . KdpD is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE . Here, we demonstrate that the phosphatase activity of KdpD is dependent on ATP, whereas GTP, ITP, CTP, ADP, and GDP have no effect . The phosphatase activity requires only ATP binding, because nonhydrolyzable analogs (adenosine-5'-{gamma-thio}triphosphate and adenosine-5'-{beta,gamma-imido}triphosphate) work as well . However, KdpD proteins missing amino acids 12-128 are characterized by a phosphatase activity that is independent of ATP . These proteins are still able to respond to K+ starvation, but an increase in osmolarity is no longer sensed . Comparison of different KdpD sequences reveals a conserved motif in this amino acid region that is very similar to a classical ATP-binding site (Walker A motif) . Replacement of the conserved Gly37, Lys38, and Thr39 residues in the consensus ATP-binding sequence results in a KdpD protein that causes a kdpFABC expression pattern comparable with that seen with KdpD proteins missing amino acids 12-128 . However, in vitro phosphatase activity is comparable with that of wild-type KdpD . These results suggest that amino acids 12-128 of KdpD are important for its activity and that an additional ATP-binding site in the N-terminal region seems to be involved in modulation of the phosphatase activity. Microb Drug Resist, 1998 Summer, 4(2), 91 - 7 Characterization of a mutationally altered dihydropteroate synthase contributing to sulfathiazole resistance in Escherichia coli; Vedantam G et al.; A series of Escherichia coli strains were selected for increasing resistance to sulfathiazole . Resistance occurred in seven increments, suggesting the accumulation of several mutations that contributed to overall sulfathiazole resistance . All of the resistant strains had a sulfathiazole-resistant dihydropteroate synthase with a Pro to Ser substitution at amino acid position 64 . Overproduction of the wild-type enzyme did not result in sulfathiazole resistance, however overproduction of the mutant enzyme resulted in significant resistance . Conversely, overproduction of the wild-type enzyme in a sulfathiazole-resistant background resulted in a decrease in resistance . Although the specific activity of DHPS in crude extracts was not significantly different from the wild type, the amino acid substitution resulted in an enzyme with a tenfold increase in the Km for p-aminobenzoate, and a 100-fold increase in the Ki for sulfathiazole. Br J Rheumatol, 1998 May, 37(5), 562 - 9 Inflammatory cytokines and cytokine antagonists in whole blood cultures of patients with systemic juvenile chronic arthritis; Muller K et al.; In the present study, we investigated the kinetics and the activation thresholds for the production of a number of pro-inflammatory cytokines and cytokine antagonists in Escherichia coli lipopolysaccharide (LPS) or phytohaemagglutinin (PHA) stimulated whole blood cultures of 13 patients with systemic juvenile chronic arthritis (SJCA) and 10 healthy children . In unstimulated cultures, the levels of interleukin (IL)-1beta, IL-6 and tumour necrosis factor alpha (TNF-alpha) were undetectable in both groups, suggesting that there was no spontaneous production of these cytokines by circulating leucocytes . The activation thresholds for the production of these cytokines, as well as the capacity for production, did not differ significantly between patients and controls . The level of interleukin-1 receptor antagonist (IL-1ra) in plasma of the patients was significantly elevated, while the in vitro production of IL-1ra was essentially normal and it did not correlate with plasma levels of IL-1ra . Supernatant levels of soluble TNF-alpha receptor (sTNF-R) I and II were both significantly elevated and correlated with the global activity score . In contrast, the supernatant levels of IL-10 were reduced in both PHA- and LPS-driven cultures . Although IL-10 levels did not correlate with laboratory or clinical indices of disease activity, the results suggest that reduced IL-10 production may play a pathogenetic role in SJCA. J Clin Microbiol, 1998 Jul, 36(7), 2099 - 102 Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis; Pacheco AB et al.; The genetic relatedness among 29 enterotoxigenic Escherichia coli strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis . The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT . Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common . These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group. Eur J Cell Biol, 1998 May, 76(1), 1 - 8 An alpha-actinin-profilin chimaera with two alternatively operating actin-binding sites; Schluter K et al.; Studying the mode of interaction between actin and actin-binding proteins, we constructed a chimaeric protein consisting of the sequence for bovine profilin I (P), to which the sequence for the actin-binding domain of Dictyostelium discoideum alpha-actinin (alphaA1-2) was fused N-terminally . The resulting hybrid clone was expressed in Escherichia coli, and the chimaeric protein, alphaA1-2P, purified by affinity chromatography on poly-(L-proline) (PLP) columns and identified using specific antibodies . High resolution electron microscopy demonstrated that this protein consists of two discrete subdomains . In biochemical, viscometric and electron microscopic analyses, we showed that both modules in this molecule are biologically active . The chimaera binds to poly-(L-proline) and inhibits the polymerization of G-actin in KCl, which is consistent with the assumption that the profilin part is intact . Inhibition of actin polymerization in KCl was stronger than that of the parental profilin, and the Kd value of its interaction with rabbit skeletal muscle actin, as determined by falling ball viscometry, was smaller (mean value 0.5 x 10(-6) M, as compared to 1.9 x 10(-6) M for bovine profilin) . In 2mM MgCl2, the actin polymerized rapidly, consistent with the interpretation that under these conditions the chimaera, like profilin, is less efficient as an actin-sequestering agent . In the presence of alphaA1-2P, the resulting filaments were decorated with particles projecting from the filament axis . We conclude that under these conditions the alphaA1-2 domain of alphaA1-2P is preferentially active, attaching the chimaeric particles laterally to the filaments . Hence, the parental modules combined in alphaA1-2P permit this molecule to switch from a G-actin- to an F-actin-binding form. FEBS Lett, 1998 Jun 12, 429(2), 201 - 6 Identification of an uncoupling mutation affecting the b subunit of F1F0 ATP synthase in Escherichia coli; Caviston TL et al.; A specific b subunit arginine, b(Arg-36) in Escherichia coli, displays evolutionary conservation among bacterial F1F0 ATP synthases . Site-directed mutagenesis was used to generate a collection of mutations affecting b(Arg-36) . The phenotype differed depending upon the substitution, and the b(Arg-36-Glu) and b(Arg-36-Ile) substitutions virtually abolished enzyme function . Although the total amounts of F1F0 ATP synthase present in the membranes prepared from mutant strains were reduced, the primary effect of the b(Arg-36) substitutions was on the activities of the intact enzyme complexes . The most interesting result was that the b(Arg-36-Glu) substitution results in the uncoupling of a functional F0 from F1 ATP hydrolysis activity. FEBS Lett, 1998 Jun 12, 429(2), 189 - 93 A G.U base pair in the eukaryotic selenocysteine tRNA is important for interaction with SePF, the putative selenocysteine-specific elongation factor; Mizutani T et al.; In Escherichia coli, selenocysteine biosynthesis and incorporation into selenoproteins requires the action of four gene products, including the specialized selenocysteine tRNA(Sec) and elongation factor SELB, different from the universal EF-Tu . In this regard, the situation is less clear in eukaryotes, but we previously reported the existence of SePF, a putative SELB homologue . The secondary structure of the tRNA(Sec) differs slightly in eukaryotes, due to a change in the lengths of several stems . Two non-Watson-Crick base pairs, G5a x U67b and U6 x U67, reside in the acceptor stem and are conserved in the course of evolution . Since it has already been reported that changing them to Watson-Crick base pairs did not affect the serylation or selenylation levels of tRNA(Sec), we asked whether these non-Watson-Crick base pairs are required for the interaction with SePF . To this end, tRNA(Sec) variants carrying Watson-Crick changes at these positions were tested for their ability to maintain the interaction with SePF . In these assays, the tRNA(Sec)-SePF interaction was determined by the protective action it confers against hydrolysis of the amino acid ester bond, under basic conditions . All the changes introduced at U6 x U67 did not significantly affect the interaction . Interestingly, however, the G5a x U67b to G5a-C67b substitution was sufficient, by itself, to lead to unprotection of the ester bond . Therefore, our finding strongly suggests that SePF is unable to interact with a tRNA(Sec) mutant version carrying a Watson-Crick G5a-C67b instead of the wild-type G5a x U67b base pair, establishing that G5a x U67b constitutes a structural determinant for SePF interaction. J Autoimmun, 1998 Apr, 11(2), 191 - 203 Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor; Diethelm-Okita B et al.; We investigated the suitability of pools of overlapping synthetic peptides spanning the complete alpha 1 subunit sequence of the human muscle acetylcholine receptor (AChR) (alpha 1 pool) or the extracellular domain (residues 1-218, alpha 11-218 pool), and of biosynthetic alpha 1 constructs from E . coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects . A construct corresponding to residues alpha 11-209 was obtained as solubilized inclusion bodies (ib alpha 11-209), or purified by SDS gel electrophoresis (pur alpha 11-209) . A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ib alpha 1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pur alpha 1NoTrans) . A biosynthetic extracellular domain of the neuronal AChR alpha 7 subunit (ib alpha 71-206) isolated from E . coli as inclusion bodies served as control for bacterial contaminants . We used ib alpha 11-209, pur alpha 11-209 and peptide pools to propagate CD4+ lines from two MG patients . The lines obtained using pur alpha 11-209 and the peptide pools recognized the peptide pools and alpha 1 constructs tested well, but ib alpha 71-206 poorly or not at all . These lines recognized peptides known to form CD4+ epitopes in these patients . The ib alpha 11-209 lines recognized ib alpha 11-209 and ib alpha 71-206 strongly, but recognized poorly pur alpha 11-209 and the alpha 11-218 pool . We propagated T-cell lines from a healthy subject using pur alpha 11-209 and ib alpha 11-209 . The pur alpha 11-209 line recognized pur alpha 11-209 and the alpha 11-218 pool, but not ib alpha 11-209 or ib alpha 71-206 . The ib alpha 11-209 line recognized ib alpha 11-209 and ib alpha 71-206, but not pur alpha 11-209 or the alpha 11-218 pool . We tested blood CD4+ cells from six MG patients and eight healthy subjects with ib alpha 11-209, pur alpha 11-209, the alpha 11-218 pool and--in the healthy subjects--also ib alpha 71-206, ib alpha 1NoTrans and pur alpha 1NoTrans . In both populations, the alpha 11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for ib alpha 11-209 than pur alpha 11-209 . The responses to ib alpha 71-206 were strong and comparable to those to ib alpha 11-209, ib alpha 1NoTrans, and pur alpha 1NoTrans . These results indicate that even purified constructs from E . coli contain bacterial contaminants recognized by CD4+ cells . They should not be used to test unselected blood CD4+ cells, because they may evoke strong CD4+ responses to the bacterial antigens . Purified recombinant sequences may be suitable for propagation of CD4+ cell lines, if the specificity of the lines can be verified using different antigen preparations . Short synthetic peptide sequences can be safely used for propagation of specific CD4+ cells . Although they are poor stimulants for unselected blood CD4+ cells, the low responses they elicit are probably due to these cells. Nucleic Acids Res, 1998 Jul 15, 26(14), 3424 - 32 Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis; Martin-Parras L et al.; Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts . To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed . The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent . Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs . Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells. Nucleic Acids Res, 1998 Jul 15, 26(14), 3364 - 71 Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence; Boybek A et al.; Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand . The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage . Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome . We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S . lividans mutants . In contrast to the pIJ101 site, each of the ADS5 . 7sites is intragenic and modified with a 10-fold reduced frequency . However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication . Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences . Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity. Genetics, 1998 Jul, 149(3), 1353 - 62 Molecular evolution of a sex determination protein . FEM-2 (pp2c) in Caenorhabditis; Hansen D et al.; Somatic sex determination in Caenorhabditis elegans involves a signal transduction pathway linking a membrane receptor to a transcription factor . The fem-2 gene is central to this pathway, producing a protein phosphatase (FEM-2) of the type 2C (PP2C) . FEM-2 contains a long amino terminus that is absent in canonical PP2C enzymes . The function of this domain is difficult to predict, since it shows no sequence similarity to any other known proteins or motifs . Here we report the cloning of the fem-2 homologue from Caenorhabditis briggsae (Cb-fem-2) . The sequence identity is much higher than that observed for other C . briggsae homologues of C . elegans sex determination proteins . However, this level is not uniform across the entire lengths of the proteins; it is much lower in the amino termini . Thus, the two domains of the same protein are evolving at different rates, suggesting that they have different functional constraints . Consistent with this, Cb-FEM-2 is able to replace some, but not all, of the Ce-FEM-2 in vivo function . We show that removal of the amino terminus from Ce-FEM-2 has no effect on its in vitro phosphatase activity, or its ability to replace the in vivo function of a yeast PP2C enzyme, but that it is necessary for proper FEM-2 function in worms . This demonstrates that the amino terminus is not an extended catalytic domain or a direct negative regulator of phosphatase activity. Genetics, 1998 Jul, 149(3), 1173 - 81 UV light induces IS10 transposition in Escherichia coli; Eichenbaum Z et al.; A new mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid was used to investigate the effect of UV light on intermolecular transposition of IS10 . Inactivation of the target gene by IS10 insertion was detected by the expression of the tet gene from the phage 434 PR promoter, followed by Southern blot analysis of plasmids isolated from TetR colonies . UV irradiation of cells harboring the target plasmid and a donor plasmid carrying an IS10 element led to an increase of up to 28-fold in IS10 transposition . Each UV-induced transposition of IS10 was accompanied by fusion of the donor and acceptor plasmid into a cointegrate structure, due to coupled homologous recombination at the insertion site, similar to the situation in spontaneous IS10 transposition . UV radiation also induced transposition of IS10 from the chromosome to the target plasmid, leading almost exclusively to the integration of the target plasmid into the chromosome . UV induction of IS10 transposition did not depend on the umuC and uvrA gene product, but it was not observed in lexA3 and DeltarecA strains, indicating that the SOS stress response is involved in regulating UV-induced transposition . IS10 transposition, known to increase the fitness of Escherichia coli, may have been recruited under the SOS response to assist in increasing cell survival under hostile environmental conditions . To our knowledge, this is the first report on the induction of transposition by a DNA-damaging agent and the SOS stress response in bacteria. Gastroenterology, 1998 Jul, 115(1), 139 - 46 Three-dimensional structure of the major autoantigen in primary biliary cirrhosis; Howard MJ et al.; BACKGROUND & AIMS: Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in patients' serum . The major autoantigen, recognized by antibodies from > 95% of patients with PBC, has been identified as the E2 component (E2p) of the pyruvate dehydrogenase multienzyme complex . Immunodominant sites on E2p have been localized to the inner of the two lipoyl domains, where the essential cofactor lipoic acid is attached covalently . The aim of this study was to determine the three-dimensional structure of the inner lipoyl domain of human E2p . METHODS: The domain was expressed in Escherichia coli; after purification, its structure was analyzed using nuclear magnetic resonance spectroscopy . RESULTS: The structure of the lipoyl domain from human E2p was determined, and the implications of the structure for autoimmune recognition were assessed . CONCLUSIONS: Knowledge of the structure further defines the major epitope and may help in the design of antigen-specific immunotherapy for treatment of PBC. EMBO J, 1998 Jul 1, 17(13), 3631 - 9 PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation; van der Wolk JP et al.; In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase . This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain . PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence . Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence . Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY . At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase . The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation . It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex. EMBO J, 1998 Jul 1, 17(13), 3512 - 20 Potent enzyme inhibitors derived from dromedary heavy-chain antibodies; Lauwereys M et al.; Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies . After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream . In contrast, the conventional antibodies apparently do not interact with the enzyme's active site . Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector . By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes . In addition we show that among those isolated fragments active site binders are well represented . When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays . The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors. J Cell Physiol, 1998 Aug, 176(2), 281 - 92 CD77-dependent retrograde transport of CD19 to the nuclear membrane: functional relationship between CD77 and CD19 during germinal center B-cell apoptosis; Khine AA et al.; A region of the N-terminal extracellular domain of the B-cell restricted cell differentiation antigen, CD19, has high amino acid sequence similarity to the receptor binding subunit B of verotoxin 1 (VT), an Escherichia coli elaborated cytotoxin, which specifically binds to the cell surface glycolipid, globotriaosylceramide, also known as the germinal center (GC) B-cell differentiation antigen, CD77 . We have previously provided evidence of the association of CD19 and CD77 on the cell surface and in CD19-mediated homotypic adhesion of the Daudi Burkitt Lymphoma cell line, one normal counterpart of which is a subset of GC B cells . Evidence for the role of CD77 in CD19-induced apoptosis is now presented . Initial cell surface distribution, antibody-induced redistribution, internalization, and intracellular routing of CD19 were studied by confocal microscopy, IF, and postembedding IEM in CD77+ve and CD77-ve cells to investigate the possible role of CD77 in CD19 internalization and signaling . Daudi Burkitt's lymphoma cells were used as CD77+ve cells and as CD77-ve cells, Daudi mutant VT500 cells, and Daudi cells treated with PPMP, an inhibitor of CD77 synthesis, were used . Antibody ligated CD19 surface redistribution, internalization, and subcellular distribution of internalized CD19 was found to be different in CD77+ve and CD77-ve cells . A delay in internalization of antibody-CD19 complex was observed in CD77-ve cells . Internalized CD19 was targeted to the nuclear envelope in CD77+ve cells in a manner similar to that reported for VT, but not in CD77-ve cells . Internalization of CD77 by ligation with verotoxin prevented the internalization of ligated CD19 . Induction of apoptosis following crosslinking of cell surface CD19 was greater in CD77+ve cells than in CD77-ve cells . The nuclear targeting of internalized CD19 and induction of apoptosis following CD19 crosslinking only in CD77+ve cells indicates a role for CD77-dependent CD19 retrograde transport from the B cell surface via the ER to the nuclear envelope in CD19-mediated signal transduction for apoptosis. Biophys J, 1998 Jul, 75(1), 453 - 62 Reversible stalling of transcription elongation complexes by high pressure; Erijman L et al.; We have investigated the effect of high hydrostatic pressure on the stability of RNA polymerase molecules during transcription . RNA polymerase molecules participating in stalled or active ternary transcribing complexes do not dissociate from the template DNA and nascent RNA at pressures up to 180 MPa . A lower limit for the free energy of stabilization of an elongating ternary complex relative to the quaternary structure of the free RNAP molecules is estimated to be 20 kcal/mol . The rate of elongation decreases at high pressure; transcription completely halts at sufficiently high pressure . The overall rate of elongation has an apparent activation volume (DeltaVdouble dagger) of 55-65 ml . mol-1 (at 35 degrees C) . The pressure-stalled transcripts are stable and resume elongation at the prepressure rate upon decompression . The efficiency of termination decreases at the rho-independent terminator tR2 after the transcription reaction has been exposed to high pressure . This suggests that high pressure modifies the ternary complex such that termination is affected in a manner different from that of elongation . The solvent and temperature dependence of the pressure-induced inhibition show evidence for major conformational changes in the core polymerase enzyme during RNA synthesis . It is proposed that the inhibition of the elongation phase of the transcription reaction at elevated pressures is related to a reduction of the partial specific volume of the RNA polymerase molecule; under high pressure, the RNA polymerase molecule does not have the necessary structural flexibility required for the protein to translocate. Biochemistry, 1998 Jun 30, 37(26), 9445 - 8 Fourier transform infrared evidence against Asp beta 99 protonation in hemoglobin: nature of the Tyr alpha 42-Asp beta 99 quaternary H-bond; Hu X et al.; The Tyr alpha 42-Asp beta 99 intersubunit H-bond stabilizes the T quaternary structure in hemoglobin (Hb) tetramers . We had proposed that Tyr alpha 42 acts as an acceptor in this H-bond, because the tyrosine Y8a/8b and Y7a' UVRR (ultraviolet resonance Raman) bands shift in directions opposite to those expected if tyrosine is an H-bond donor . If Asp beta 99 is the H-bond donor, then it must be protonated in the T state, and would be a previously unrecognized contributor to the Bohr effect . This implication was strengthened by the discovery that an R-minus-T difference FTIR (Fourier transform infrared) band at 1693 cm-1, which might be a signal from protonated carboxylate, is missing in Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn . However, we now find that this FTIR signal is insensitive to 13C-labeling of the aspartate residues in Hb, and cannot arise from protonated Asp beta 99 . There are no other difference signals in the 1700 cm-1 region at a sensitivity of one COOH group . We conclude that Asp beta 99 is not protonated, and that the anomalous UVRR shifts must arise from compensating polarization of the Tyr alpha 42 OH . Candidates for this compensation are the H-bond donated by the Asp beta 94 backbone NH, and the nearby positive charge of Arg beta 40. Clin Exp Immunol, 1998 May, 112(2), 255 - 61 Genetic identification of antigens exposed in damaged endothelial cells as laminin-binding proteins; Ireland DC et al.; A monoclonal antibody, D5G2, which reacts in a balloon angioplasty damage model with unfixed damaged but not with unfixed undamaged human endothelial cells, was used to screen a human endothelial cDNA library in an Escherichia coli/lambda gt11 expression system . Sequences of DNA inserts in D5G2+ phage clones matched those reported for a laminin-binding protein, LBP-32 . Both D5G2 and purified laminin bound to a polypeptide of 55 kD on PVDF membranes carrying electrophoretically separated endothelial cell lysates, D5G2 also bound to recombinant LBP expressed in E . coli, and showed similar staining patterns on human and bovine endothelial cells to another characterized anti-LBP antibody . Increased staining of unfixed endothelial cells on detergent permeabilization suggests that D5G2 binds to intracellular laminin-binding protein made accessible by cell membrane injury . Antibodies to intracellular targets exposed by cell damage may be useful in anchoring therapeutic agents at sites of vascular damage. Mol Gen Genet, 1998 May, 258(4), 442 - 7 In vitro and in vivo characterization of three major dadAX promoters in Escherichia coli that are regulated by cyclic AMP-CRP and Lrp; Zhi J et al.; We have shown previously that the dadAX operon of Escherichia coli expresses multiple transcripts, which are repressed by leucine-responsive regulatory protein (Lrp) . Here we used site-directed mutagenesis and in vitro and in vivo transcription assays to show that each of the three major dad transcripts requires a specific promoter . These promoters, P1-P3, overlap and are positively regulated in vivo by cyclic AMP-CRP . DNase I footprinting experiments localized two CRP binding sites in this region: CRP1, which is positioned upstream of P1-P3, and CRP2, which is located within the promoters . Site-directed mutagenesis of each site provided evidence that CRP1 is necessary for the effects of cyclic AMP-CRP on dad expression in vivo and in vitro, and that CRP2 probably plays little or no role in this process. Mol Gen Genet, 1998 May, 258(4), 404 - 11 The uvp1 gene on the R46 plasmid encodes a resolvase that catalyzes site-specific resolution involving the 5'-conserved segment of the adjacent integron In1; Tosini F et al.; The product of the uvp1 gene of the R46 plasmid, a member of the DNA invertase-resolvase family, was studied to characterize its recombination activity on the R46 plasmid . The purified Uvp1 protein specifically binds to a 256-bp DNA fragment located immediately upstream of the uvp1 gene itself, and overlapping the 5'-conserved segment (5'-CS) of the R46 integron In1 . We identified on this fragment a putative resolution (res) site . Using an in vitro assay, we demonstrated the ability of the protein to resolve a synthetic cointegrate containing a direct repeat of the res site . In vivo, we obtained cointegrate resolution in Uvp1-expressing recA- cells . Sites I and II, subsites of the putative res site, lie within the outer boundary of the integron 5'-CS which is common to all the known integrons . Furthermore, a 69-bp DNA element (containing site I) is required for cointegrate resolution . We propose that this recombination mechanism protects R46 plasmid against unequal distribution following fusion with either identical or different integron-bearing plasmids . Moreover, Uvp1 might have a role in generating gene cassette diversity between the two conserved segments of the integron. J Allergy Clin Immunol, 1998 Jun, 101(6 Pt 1), 832 - 40 Cloning of the American cockroach Cr-PII allergens: evidence for the existence of cross-reactive allergens between species; Wu CH et al.; BACKGROUND: Previously, we have identified the 28 and 32 kd proteins as additional important allergens from the American cockroach (Periplaneta americana) Cr-PII allergenic fraction . OBJECTIVE: The aim of this study was the cloning of P . americana Cr-PII allergens . METHODS: A lambdagt22A cDNA library constructed from P . americana mRNA was packaged into Escherichia coli Y1090 (r-), and clones recognized by murine anti-Cr-PII monoclonal antibodies and human IgE antibodies were isolated, sequenced, and subcloned into pET 21 and expressed in E . coli BL21(DE3) . RESULTS: Six Cr-PII-positive clones recognized by human IgE antibodies were isolated . Two clones, C6 and C17, were sequenced, and we found encoding proteins of 228 and 274 amino acids with no cysteine or any potential N-glycosylation site, with predicted masses of 25.8 and 31.14 kd, respectively . Both molecules contain internal repeated sequences with a 94% identity between them . C6 and C17 showed 59% and 77.3% skin reactivities, respectively, on 22 cockroach-sensitive atopic patients . Both clones were found to have 28.9% to 31.8% identities to ANG12 protein, a precursor of the African malaria mosquito (Anopheles gambiae) and 82.7% to 85.1% identity to a nucleotide sequence of the German cockroach (Blattella germanica) Bla g Bd90K allergen . The anti-C6 and anti-C17 antibodies were able to recognize Cr-PII, recombinant proteins, five commercial American extracts, and two German cockroach extracts . Moreover, the binding of anti-C6 and anti-C17 antibodies to recombinant protein can be inhibited by B . germanica crude extract . Furthermore, Northern blot analyses have shown that B . germanica mRNAs could be detected by both cDNA probes . CONCLUSION: Our findings provide the first evidence of antigenic cross-reactivity between P . americana and B . germanica allergens on molecular levels . The results will be a great aid in facilitating the epitope mapping and improving diagnostic and therapeutic reagents for both cockroach species. J Allergy Clin Immunol, 1998 Jun, 101(6 Pt 1), 772 - 7 Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen; Tinghino R et al.; BACKGROUND: Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries . OBJECTIVE: We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupressaceae family . METHODS: Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector . IgE screening of the library was performed with a pool of sera from subjects allergic to Cupressaceae . A recombinant 6xHis-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni2+ affinity chromatography . It was studied further by immunoblotting inhibition with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae . The role of protein-bound calcium on the allergen's IgE-binding capacity was tested in a plaque assay in the presence or absence of EGTA . RESULTS: A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated . The deduced amino acid sequence contained four typical Ca2+ binding sites and showed a significant sequence similarity to calmodulins . Depletion of Ca2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2 . Immunoblotting inhibition revealed that J . oxycedrus, J . ashei, Cupressus arizonica, C . sempervirens, Parietaria judaica, Olea europaea, and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations . CONCLUSION: rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen . These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered. Anal Biochem, 1998 Jun 15, 260(1), 18 - 23 Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells; Ernst JD et al.; Annexin proteins specifically bind anionic phospholipids such as phosphatidylserine, which are normally confined to the cytoplasmic leaflet of cellular membranes . During programmed cell death, or apoptosis, this phospholipid asymmetry is lost, and anionic phospholipids are exposed on the extracellular leaflet of the plasma membrane where they are accessible to exogenously added, labeled annexins . Chemically {e.g., fluoroscein isothiocyanate (FITC)}-modified annexin V has been widely used to detect and enumerate apoptotic cells by flow cytometry . We prepared chimeric proteins containing green fluorescent protein (GFP) fused to annexin V . A chimera containing GFP fused to the C-terminus of annexin V was soluble and fluorescent, but was unable to bind phospholipids . In contrast, a chimera containing GFP fused to the N-terminus of annexin V specifically bound apoptotic cells . GFP-annexin V represents a sensitive and facile alternative to FITC-annexin V for studies of apoptosis. Yi Chuan Xue Bao, 1998, 25(1), 86 - 94 {Study on the induction of pco promoters from Escherichia coli with copper and other metal ions}; Liu ZP et al.; Two copper inducible promoters in the pco determinant of Escherichia coli were studied by determining the luciferase activity of report vector pUCD615 . The results showed that in the absence of pBIN19pco, providing copper resistance genes in trans, the maximum induction for both PpcoA lux fusions was observed at 5 mmol/L CuSO4 and PpcoA box was a stronger promoter than PpcoA long . There were two peaks in the bioluminescences of both PpcoE-lux fusions induced with increasing copper concentration, the first peak was observed at about 0.5 mmol/L CuSO4, the second peak, also the maximum induction, was observed at about 5 mmol/L CuSO4, and PpcoE long was a stronger promoter than PpcoE box . The results indicated that the PpcoE promoter was a much stronger promoter than PpcoA promoter . The results also showed that the copper box was very important and essential to pco promoters, since both of the Ppco short-lux fusions failed to show any luciferase activity when they were induced with copper . In the presence of pBIN19pco, the maximum inductions of all of the Ppco-lux fragments were observed at 6 mmol/L CuSO4 and they were much higher than those observed in the absence of pBIN19pco, and the results also indicated that the cells were able to resistant to much higher copper concentration in the presence of pBIN19pco than in the absence of that . Zn2+ and Ni2+ could be inducers for all of the fragments and Zn2+ was a better inducer than Ni2+, and Cd2+ and Ag+ did not induce the pco system. Carbohydr Res, 1997 Dec, 305(3-4), 533 - 41 The chemoenzymatic synthesis of the core trisaccharide of N-linked oligosaccharides using a recombinant beta-mannosyltransferase; Watt GM et al.; The chemical synthesis of the beta-mannosyl linkage of N-glycans has presented a great challenge to synthetic carbohydrate chemists . We have therefore investigated the application of beta-mannosyltransferases to the preparative synthesis N-linked core oligosaccharides . In this paper we report the chemoenzymatic synthesis of beta-D-mannopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose on a preparative scale using a phytanyl-linked acceptor in the presence of a recombinant beta-(1-->4)-mannosyltransferase. Carbohydr Res, 1997 Dec, 305(3-4), 383 - 91 Regioselectivity of the enzymatic transgalactosidation of D- and L-xylose catalysed by beta-galactosidases; Montero E et al.; The regioselectivity of enzymatic transgalactosidation depends on the source of the beta-galactosidase used . When the galactosyl acceptor only contains secondary hydroxyl groups, e.g., D- or L-xylose, it is possible to find an enzyme that catalyses preferentially the synthesis of any of the three regioisomers 4-, 3- and 2-O-beta-D-galactopyranosyl-D-xylose (1, 2 and 3, respectively) or 4-, 3- and 2-O-beta-D-galactopyranosyl-L-xylose (4, 5 and 6, respectively) . Enriched mixtures in 1, 2 or 3 were obtained using beta-galactosidases from Escherichia coli, bovine testes or Aspergillus oryzae, respectively, by transgalactosidation reaction of O-nitrophenyl-beta-D-galactopyranoside and D-xylose, and enriched mixtures in 4, 5 or 6 were obtained in a similar way using beta-galactosidases from Aspergillus oryzae, lamb small-intestine (intestinal lactase-phloridzin hydrolase) or Saccharomyces fragilis, respectively, using L-xylose as acceptor. Biosci Biotechnol Biochem, 1998 May, 62(5), 870 - 4 Cloning and characterization of the azurin iso-1 gene, concerned with the electron transport chain involved in methylamine/methanol oxidation in the obligate methylotroph Methylomonas sp . strain J; Taguchi K et al.; Two azurin-type blue copper proteins, which is concerned with the electron transport chain involved in methylamine/methanol oxidation, have been found in the obligate methylotroph Methylomonas sp . strain J . The azurin iso-1 gene was cloned and sequenced to analyze the role in the electron transport chain . PCR products synthesized with primers based on the N- and C-terminal amino acid sequences of azurin iso-1 were used as probes for cloning . One complete open reading frame (the azurin iso-1 gene) and one partial orf (orf1) were found in a cloned Eco105I-HindIII fragment, pMAZ3, with a total of 1066 bp . The gene encoded 148 amino acid residues . The amino acid sequence after Ala-21, deduced from the nucleotide sequence, was identical to that of the azurin iso-1 protein . The gene was in a region separate from the mau gene cluster in the chromosome . Escherichia coli expressed azurin iso-1 . The results of northern blotting analysis suggested that expression of the azurin iso-1 gene is regulated by a complex regulatory network controlling oxidation of methylamine or methanol in this strain; for example, copper ions affected the expression of the azurin iso-1 gene. Appl Environ Microbiol, 1998 Jul, 64(7), 2609 - 15 Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme; Chen L et al.; Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1'-phosphate, an osmolyte found in hyperthermophilic archaeal . Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity . The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K(m) = 0.091 +/- 0.016 mM; Vmax = 9.3 +/- 0.45 mumol of Pi min-1 mg of protein-1) comparable to those of mammalian and E . coli enzymes . Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases . The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources . The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M . jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme. Appl Environ Microbiol, 1998 Jul, 64(7), 2601 - 8 phnE and glpT genes enhance utilization of organophosphates in Escherichia coli K-12; Elashvili I et al.; Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (< or = 1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources . Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate . A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation . These genes were identified as phnE and glpT . The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E . coli K-12 phnE gene that has been shown previously to result in phnE activation . In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation . PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid . This suggests that the OPU phenotype associated with the glpT gene is the result of amplification and overproduction of the glpT gene product . Both the active phnE and multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnE gene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-Pi) . This result indicates that in E . coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems. Appl Environ Microbiol, 1998 Jul, 64(7), 2357 - 60 Thermotoga neapolitana homotetrameric xylose isomerase is expressed as a catalytically active and thermostable dimer in Escherichia coli; Hess JM et al.; The xylA gene from Thermotoga neapolitana 5068 was expressed in Escherichia coli . Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form . The purified native enzyme, however, has been shown to be exclusively tetrameric . The two enzyme forms had comparable stabilities when they were thermoinactivated at 95 degrees C . Differential scanning calorimetry revealed thermal transitions at 99 and 109.5 degrees C for both forms, with an additional shoulder at 91 degrees C for the tetramer . These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme. Biochem Biophys Res Commun, 1998 Jun 29, 247(3), 554 - 7 Replication of lambda plasmid DNA in the Escherichia coli cell cycle; Herman-Antosiewicz A et al.; The Cro repressor autoregulatory loop has long been considered the main regulatory process in controlling lambda plasmid replication initiation in Escherichia coli . However, we found recently that lambda plasmids can be maintained at a constant copy number in the absence of Cro function . Here we demonstrate that shortly after inactivation of the Cro repressor, the synthesis of lambda plasmid DNA increases significantly but is then stabilized at a level similar to that observed in the presence of the Cro function . We found that replication initiation of lambda plasmids carrying a functional cro gene proceeds randomly in the host cell cycle, but in the absence of Cro function the replication initiation of lambda plasmid DNA appears to be cell cycle dependent . The host DnaA protein appears to be at least one of the factors involved in the cell-cycle-specific control of lambda cro- plasmid replication . Therefore, it seems that the lambda cro- plasmid may serve as an amazingly simple model for studies on the regulation of DNA replication in the cell cycle. J Invertebr Pathol, 1998 Jul, 72(1), 44 - 9 Chemotactic responses of tunicate (Urochordata, Ascidiacea) hemocytes in vitro; Raftos DA et al.; A number of molecules were found to alter the motility of tunicate hemocytes . Bacterial lipopolysaccharide (LPS) significantly enhanced cell mobility relative to non-stimulated controls . Responses to LPS were not directional and so represented chemokinesis . In contrast, checkerboard analyses indicated that two tunicate hemolymph proteins, tunIL1-alpha and -beta, stimulated truly directional chemotaxis by hemocytes . The data suggest that tunIL1 proteins may contribute to defense by altering the localization of immunocompetent cells . J Cell Biol, 1998 Jun 29, 141(7), 1529 - 37 G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton; Peracino B et al.; Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae . Deletion of the unique G protein beta subunit in D . discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility . Constitutive expression of wild-type beta subunit restored phagocytosis and normal development . Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells . In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis . Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment . Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis . Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process. Hum Reprod, 1998 May, 13(5), 1175 - 9 Circulating antibodies to a conserved epitope of the Chlamydia trachomatis 60 kDa heat shock protein (hsp60) in infertile couples and its relationship to antibodies to C.trachomatis surface antigens and the Escherichia coli and human HSP60; Witkin SS et al.; To evaluate the relationship between immunity to specific regions of the Chlamydia trachomatis 60 kDa heat shock protein (hsp60), autoimmunity to human HSP60 and infertility, sera from 50 women and 45 men seen for an infertility evaluation were tested . Humoral immunity to human HSP60 was detected in 18% of women and 8.9% of men while antibodies to the Escherichia coli hsp60 were detected in 12% of women and 4.4% of men . These differences were not statistically significant . In contrast, antibodies to a synthetic peptide epitope of the chlamydial hsp60, encompassing amino acids 260-271 (chsp 260-271), were present in sera from 16 (32%) of the women but in only six (13.3%) of the men (P=0.03) . Antibodies to chsp 260-271 were present in 11 out of 17 (64.7%) individuals with high titre (>1:160) immunoglobulin (Ig)G antibody to C.trachomatis surface antigens as opposed to only two out of 15 (13.3%) with low titre antibody and two out of of 17 (11.8%) with undetectable chlamydial antibody (P < 0.004) . Antibodies to chsp 260-271 were also associated with humoral immunity to human HSP60 . 50% of sera with, as opposed to only 18.6% of sera without, anti-human HSP60 IgG were positive for antibodies to chsp 260-271 (P=0.03) . In contrast, there was no relationship found between immunity to the E.coli hsp60 and antibodies to human HSP60 . Antibodies to chsp 260-271 were more prevalent in women with at least two spontaneous abortions (eight out of 13, 61.5%) than in women with other infertility diagnoses (six out of 35, 17.1%) (P=0.004) . Thus, immunity to chsp 260-271 is more prevalent in women than in men, associated with autoimmunity to human HSP60 and may be an immunological marker for spontaneous abortion. Hum Reprod, 1998 May, 13(5), 1163 - 8 Endocytosis and MHC class II expression by human oviductal epithelium according to stage of the menstrual cycle; Imarai CM et al.; The epithelium is the first barrier against pathogens invading the lumen of the human oviduct . Its expression of class II major histocompatibility complex (MHC class II) proteins suggests that it might play a role in antigen presentation during the local immune response . To study the role of the oviductal epithelium in antigen processing, its endocytic properties and MHC class II expression were examined . For assay of endocytosis, fluorescein isothiocyanate-labelled bovine serum albumin (BSA-FITC) or Escherichia coli (E . coli-FITC) was infused into the lumen . One-centimetre pieces of oviduct were incubated for 2 h and processed for fluorescence and confocal microscopy, and transmission electron microscopy . Incorporation into secretory and ciliated epithelial cells was observed, which was unrelated to the phase of the menstrual cycle . Small pieces of the organs were frozen and processed for immunohistochemistry . Most oviducts expressed MHC class II (HLA.DR) in the epithelium and in some cases this was coincident with endocytosis, but there was no statistically significant association between this expression and either endocytotic activity or the phase of the menstrual cycle . Results demonstrate that the epithelium of the human oviduct exhibits endocytic properties towards luminal soluble and particle antigens, which is not related either to MHC class II expression or to the phase of the menstrual cycle. Br J Pharmacol, 1998 Jun, 124(3), 586 - 92 Effects of inhibitors of the activity of cyclo-oxygenase-2 on the hypotension and multiple organ dysfunction caused by endotoxin: a comparison with dexamethasone; Leach M et al.; 1 . Endotoxaemia is associated with the expression of the inducible isoform of cyclo-oxygenase, cyclo-oxygenase-2 (COX-2), and an overproduction of arachidonic acid (AA) metabolites . The role of the AA metabolites generated by COX-2 in the circulatory failure and multiple organ dysfunction caused by endotoxin is unclear . Dexamethasone prevents the expression of COX-2 and exerts beneficial effects in animal models of shock . 2 . Here we compare the effects of two inhibitors of COX-2 activity, namely NS-398 (5 mg kg(-1), i.p., n=7) and SC-58635 (3 mg kg(-1), i.p., n=9) with those of dexamethasone (3 mg kg(-1), i.p., n=9) on the circulatory failure and organ dysfunction caused by lipopolysaccharide (LPS, E . coli, 6 mg kg(-1), i.v., n=11) in the rat . 3 . Endotoxaemia for 6 h caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of 6-keto-PGF1alpha (indicator of the induction of COX-2) and nitrite/nitrate (indicator of the induction of iNOS) . 4 . Pretreatment of rats with dexamethasone attenuated the hypotension, the renal dysfunction, the hepatocellular and pancreatic injury and the induction of COX-2 and iNOS caused by LPS . In contrast, inhibition of COX-2 activity with SC-58635 or NS-398 neither attenuated the circulatory failure nor the multiple organ failure caused by endotoxin . 5 . Thus, the prevention of the circulatory failure and the multiple organ injury/dysfunction caused by dexamethasone in the rat is not due to inhibition of the activity of COX-2 . Our results suggest that an enhanced formation of eicosanoids by COX-2 does not contribute to the development of organ injury and/or dysfunction in rats with endotoxaemia. J Immunol, 1998 Jul 1, 161(1), 448 - 57 T cell epitopes in Japanese cedar (Cryptomeria japonica) pollen allergens: choice of major T cell epitopes in Cry j 1 and Cry j 2 toward design of the peptide-based immunotherapeutics for the management of Japanese cedar pollinosis; Sone T et al.; Japanese cedar pollinosis is caused by exposure to Japanese cedar (Cryptomeria japonica) pollen, of which two components, Cry j 1 and Cry j 2, are believed to be the major allergens . T cell lines specific to either Cry j 1 or rCry j 2 were reactive to various portions of each panel of overlapping peptides derived from Cry j 1 or Cry j 2 . Two peptides, p211-225 and p108-120, from among six major T cell epitopes identified in Cry j 1 sequence, and three peptides, p182-200, p344-355, and p66-80, from among five in Cry j 2, were chosen to design an artificial polypeptide (named Cry-consensus) based on a difference among the types of the restriction molecules capable of presenting these peptides . After construction of a DNA encoding these peptides in order, Cry-consensus was expressed in Escherichia coli . Five of six T cell epitopes, except for Cry j 2 p344-355, in Cry-consensus were recognized by the T cell clones specific to each peptide . PBMC from allergic patients induced higher proliferation under stimulation from Cry-consensus than individual peptides . Eighty-eight percent of the PBMC (15 of 17) showed proliferation under the Cry-consensus stimulation . Thus, several major T cell epitopes from Cry j 1 and Cry j 2 can be chosen in the design of peptide-based immunotherapeutics for the management of Japanese cedar pollinosis in subjects having various types of HLA class II molecules. J Immunol, 1998 Jul 1, 161(1), 138 - 47 Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes; Marazzi S et al.; We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen . Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli . mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC . Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex . Because of its homology with fibrinogen, we have termed this protein fibroleukin . Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes . RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+) . Fibroleukin production by PBMC was rapidly lost in culture . Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect . To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology . Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa . While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites. Annu Rev Biophys Biomol Struct, 1998, 27, 357 - 406 The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins; Gardner KH et al.; During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of applications . Most recently, the combination of triple resonance experiments and 2H, 13C, 15N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa . Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems . As well, we detail recent progress in the use of solution 2H NMR methods to probe the dynamics of protein sidechains. Annu Rev Biophys Biomol Struct, 1998, 27, 35 - 58 The three-dimensional structure of the ribosome and its components; Moore PB; Exciting progress has been made in the last decade by those who use physical methods to study the structure of the ribosome and its components . The structures of 10 ribosomal proteins and three isolated ribosomal protein domains are known, and the conformations of a significant number of rRNA sequences have been determined . Electron microscopists have made major advances in the analysis of images of ribosomes, and microscopically derived ribosome models at resolutions approaching 10A are likely quite soon . Furthermore, ribosome crystallographers are on the verge of phasing the diffraction patterns they have had for several years, and near-atomic resolution models for entire ribosomal subunits could emerge from this source at any time . The literature relevant to these developments is reviewed below. Vet Microbiol, 1998 Mar 15, 61(1-2), 51 - 8 Binding characteristics of purified Escherichia coli K88ab fimbriae to guinea pig erythrocyte membrane; Caloca MJ et al.; The characteristics of the binding of biotinylated E . coli K88ab fimbriae to guinea pig erythrocyte membranes, as a possible model of the target host cell were studied . Binding showed sigmoidal dependence, with an apparent saturation at about 0.8 ng of fimbriae . Hill coefficient values (h) were about 2-2.4, which indicated that the receptor population showed positive cooperativity with at least three binding sites . Apparent binding constants to the first and third binding sites (1K3 and 3K3) were determined . Three K88ab binding proteins, of 67, 63 and 48 kDa, were identified on solubilized erythrocyte membranes and were recovered mainly in a detergent phase, suggesting a possible integral localization of the receptors. Vet Microbiol, 1998 Feb 28, 60(2-4), 227 - 38 An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function; Nagai S et al.; An avian pathogenic Escherichia coli strain M1000 showed a clear zone of erythrocyte lysis on sheep blood agar plates . The hemolytic activity was not detected in the culture supernatant nor was any DNA sequence homologous to the E . coli alpha-hemolysin gene detected in the chromosome or plasmid DNA of the strain, indicating that the observed hemolysis was different from alpha-type . To identify the genetic determinant responsible for the hemolysis, we performed random Tn5 insertional mutagenesis and obtained one mutant, named M5005, that totally lacked the hemolytic activity . Cloning and nucleotide sequencing of the region flanking the transposon insertion site in the M5005 chromosome revealed that the transposon was inserted within an open reading frame of the cyclic AMP receptor protein (CRP) gene, which is involved in one of the global regulatory networks of gene expression in E . coli . Nucleotide sequence analysis of the intact crp gene of the strain M1000 showed that the CRP protein of M1000 is 99% identical to that of K-12 . Introduction of the intact crp gene on a low copy plasmid into the mutant M5005 restored the hemolytic phenotype, confirming that the mutation site in M5005 was in the crp gene . CRP plays a central role in catabolite repression, the phenomenon by which the synthesis of many enzymes required to metabolize various sugars is repressed in the presence of glucose . When the hemolytic activity of E . coli M1000 grown in the presence of glucose was examined, the hemolysis was totally impaired . These results indicate that the avian pathogenic E . coli strain M1000 produces a hemolysin the expression of which is dependent on crp gene function. Vet Immunol Immunopathol, 1998 Apr 30, 62(4), 309 - 21 Compensation of preliminary blood phagocyte immaturity in the newborn calf; Menge C et al.; To estimate the functional maturity of the phagocytic defence in neonatal calves, we analyzed the characteristics of blood phagocytes from calves (n = 10) 1 h post partum (p.p.) and 4 h p.p . At 1 h p.p., all calves were colostrum-deprived, while 5 calves had received colostrum before the 4 h p.p . sampling . The results were compared to those obtained from 3-9-week-old calves (n = 10) . Phagocytic and oxidative burst activity of polymorphonuclear leukocytes (PMNL) and monocytes were determined in whole blood and separately analyzed by flow cytometry . In neonates prior to colostrum ingestion (1 h p.p.), phagocytic activity of PMNL against non-preopsonized E . coli was lower when compared to PMNL of 3-9-week-old calves . Opsonization of bacteria with pooled plasma from adult animals only partially restituted this lower PMNL phagocytic activity, indicating that humoral as well as cellular aspects of PMNL phagocytosis are altered in neonatal calves . In contrast to PMNL, monocytes of neonates exhibited an enhanced phagocytic activity . The oxidative burst activity of PMNL, as well as of monocytes was higher in newborn calves . During the first 4 h of life, the activities of blood phagocytes changed . Colostrum ingestion was accompanied by an increase in the percentage of phagocytizing PMNL and monocytes . This increase was absent in colostrum-deprived calves . In contrast, the oxidative burst activity of phagocytes decreased with age . In monocytes, the decrease of oxidative burst activity was only observed in colostrum-fed calves . In conclusion, some blood phagocyte functions in calves were found to be immature at birth, but these functions are presumably compensated by high absolute PMNL numbers and by other the more active mechanisms. Ital J Biochem, 1998 Mar, 47(1), 13 - 8 Effects of wortmannin on human neutrophil respiratory burst and phagocytosis; Santoro P et al.; Modulation of neutrophil response to naturally occurring stimuli is important to avoid host tissue injure . Both soluble and particulate stimuli may induce superoxide anion generation in human polymorphonuclear leukocytes . Recently wortannin has been shown to inhibit the N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced activation of respiratory burst via phosphatidylinositol 3-kinase . However no data are available about the effect of the inhibitor on the respiratory burst induced by a particulate stimulus . In this paper we studied the effect of wortmannin on E . coli induced respiratory burst and phagocytosis by flow cytometry, which allows the quantitation of both H2O2 production and ingested bacteria in whole blood samples without the need of purification and concomitant manipulation of the cells . The effects of worthmannin on fMLP-induced chemotaxis was also examined by the under agarose method . Neither the E . coli nor the fMLP-induced responses were blocked by wortmannin, suggesting that PI 3-kinase activity is not required to activate these neutrophil functions . Since it is known that the respiratory burst elicited by fMLP is blocked by wortmannin, our results suggest that the generation of oxygen radicals is controlled via different signal transduction pathways, depending on the agonist used. Shock, 1998 Jun, 9(6), 428 - 33 Effect of liposome-encapsulated hemoglobin on the development of endotoxin-induced shock in the rat; Whiteford M et al.; Liposome-encapsulated hemoglobin (LEH) is an experimental oxygen-carrying resuscitation fluid . Because LEH is cleared from the circulation primarily by the reticuloendothelial system, its effect on the development of sepsis remains a major concern . Thus, the present study aimed to evaluate whether LEH modifies consequences of endotoxemia in the conscious normovolemic rat . LEH infusion at 10% of estimated blood volume (n = 10) did not affect mortality (30%, p < .05) and serum tumor necrosis factor-alpha levels (6204 +/- 414, p < .05) induced by 3.6 mg/kg Escherichia coli endotoxin administered (intravenous bolus) 22 h later . In contrast, when a shorter LEH-endotoxin time interval (<12 h, n = 10) or a higher dose of endotoxin (14.4 mg/kg, n = 20) was tested, LEH enhanced endotoxin-induced mortality (90% and 100%, respectively, p < .05) and broadened serum tumor necrosis factor-alpha response without modifying its peak levels . LEH (n = 20) did not exacerbate the endotoxin-induced tachycardia, leukopenia, and thrombocytopenia . Therefore, in this model, the effect of LEH on endotoxin-induced responses was dependent on the time interval between LEH and endotoxin administration as well as the endotoxin dose . The clinical relevance of these results should be further investigated. FEBS Lett, 1998 May 22, 428(1-2), 105 - 10 Cloning and expression of the gene for a vanadium-dependent bromoperoxidase from a marine macro-alga, Corallina pilulifera; Shimonishi M et al.; The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Corallina pilulifera . The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme . The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme . From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C . pilulifera growing in a relatively narrow area . The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum . The homology search of the sequences of bromoperoxidases from C . pilulifera (this study) and A . nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD . Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C . inaequalis are conserved in the primary structure of the bromoperoxidase from C . pilulifera . The cloned hpol was introduced into Escherichia coli, and the expressed PO1 was purified from the recombinant strain . The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine. Mol Gen Genet, 1998 May, 258(3), 240 - 9 Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein; Matsuo E et al.; We previously described a dominant negative secY-d1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY . Syd is the product of a multicopy suppressor of the secY-d1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein . In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction . We isolated Syd-resistant revertants from the secY24 mutant . Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240-->Asp) . The secY249 mutation (Ala249-->Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation . The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction . The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein . Although the secY245 mutant retained some sensitivity to Syd overproduction, the secY241 mutant was completely Syd-resistant . Furthermore, the secY241 mutation seemed to represent a "hyper reversion" with respect to the SecY-SecE interaction . Protein export in this mutant was no longer sensitive to SecY-d1 . When the secY-d1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY-d1-241) showed an increased ability to interfere with protein export . On the basis of our model for SecY-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction . These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex. Avian Dis, 1998 Apr-Jun, 42(2), 285 - 91 Cellulitis in broiler chickens: epidemiological trends, meat hygiene, and possible human health implications; Kumor LW et al.; The present work evaluates trends in the incidence of cellulitis during the last decade using Canadian National Poultry Condemnation Records . In 1986, only 0.048% of the total slaughter broilers were condemned as a result of cellulitis lesions . Over the next 10 yr, steady increments in cellulitis condemnations were observed, and between 1986 and 1996, the percentage of cellulitis condemnation increased 11.8-fold . In 1996, more than 2.6 million broilers (0.568% of total slaughter) were condemned due to cellulitis; this constituted 30.1% of total condemnations, making it the number one condemnation category in 1996 . In the context of dynamic increase in cellulitis, the problems concerning meat hygiene and possible health risk to the consumer are deliberated. Arch Virol, 1998, 143(5), 963 - 70 Characterization of Trichomonas vaginalis virus proteins in the pathogenic protozoan T . vaginalis; Liu HW et al.; The 4.6-kb double-stranded (ds) RNA of Trichomonas vaginalis virus (TVV)-T1 has been shown to encode two overlapping genes, cap and pol . In this study, a serum for specifically detecting viral cap gene product was raised against a recombinant protein, and sera for specifically detecting pol gene product were raised against synthetic oligopeptides . A 75-kDa major protein and a 160-kDa minor protein were detected by anti-CAP serum in a TVV-T1 sample, indicating that the 75-kDa protein is the viral capsid protein . The 160-kDa protein alone was also detected by two distinct anti-POL sera, indicating that the pol gene is expressed as a CAP-POL fusion protein . These results suggest that the TVV-T1 genome is arranged into a cap-pol organization in a manner similar to that of viruses in family Totiviridae. Arch Virol, 1998, 143(5), 931 - 44 Membrane association and RNA binding of recombinant hepatitis A virus protein 2C; Kusov YY et al.; The direct function of hepatitis A virus (HAV) protein 2C, a putative NTPase, is not known, yet genetic evidence obtained from chimeric viruses carrying the 2C genomic region of different HAV variants indicates that it plays a pivotal role in viral replication . In a first assessment of its potential function(s), membrane and RNA binding properties of HAV 2C were studied after expressing the protein in various recombinant systems . In contrast to poliovirus 2C, expression of HAV 2C was inhibitory to the growth and protein synthesis of bacteria . Deletion of the N-terminal amphipathic helix of 2C abrogated this effect and the ability of 2C to associate with eukaryotic membranes . Both, purified 2C and the N-terminally truncated protein were shown to bind RNA in vitro . Our data taken together suggest that HAV 2C is a multifunctional protein. Arch Virol, 1998, 143(5), 863 - 80 Characterization of the UL16 gene product of herpes simplex virus type 2; Oshima S et al.; We have raised rabbit polyclonal antisera against a His-tagged herpes simplex virus type 1 (HSV-1) UL16 fusion protein, one of which very specifically reacted with 40 kDa and 41 kDa proteins in the lysates of HSV-1 and HSV-2-infected cells, respectively . Since its reactivity to the 41 kDa protein was clearly eliminated by pre-adsorption with E . coli lysates expressing the UL16 fusion protein, the antiserum was used to characterize the UL16 products of HSV-2 . The HSV-2 UL16 protein was produced at the late phase of infection in a manner highly dependent on viral DNA synthesis and was distributed in both the nuclei and the cytoplasma of infected cells . In immunofluorescence studies, the UL16-specific fluorescence in the nuclei was shown to be detected as small discrete granules . On the other hand, the cytoplasmic fluorescence was diffusely distributed around the nucleus at 8 h postinfection but, at later times of infection, it was mainly detected as a mass at a perinuclear region . The analysis on its association with capsids has revealed that the UL16 protein copurified with C capsids but not B and A capsids, and that the association with C capsids was not tight . Moreover, our experiments have shown that a detectable level of the UL16 protein was not associated with extracellular virions, and that the partially purified UL16 proteins had a DNA-binding activity . These observations are consistent with the hypothesis that the UL16 protein plays a role in capsid maturation including DNA packaging/cleavage . We have also determined the complete nucleotide sequence of the HSV-2 UL16 gene and found that a nonstandard initiation codon may be used for its translation. Prikl Biokhim Mikrobiol, 1998 May-Jun, 34(3), 293 - 9 {Biosynthesis and isolation of a recombinant protein for producing genetically-engineered human proinsulin}; Ivankin AN et al.; Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied . Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins . Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis . Human proinsulin was derived from the recombinant protein isolated. J Biochem (Tokyo), 1998 Jul, 124(1), 187 - 93 Evidence for chloramphenicol/H+ antiport in Cmr (MdfA) system of Escherichia coli and properties of the antiporter; Mine T et al.; We detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E . coli . Introduction of the gene into E . coli cells conferred resistance to chloramphenicol and ethidium . A slight increase in resistance to acridine orange was also observed . Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene . Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium . Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid . The pH optimum for the activity was 6.5 . We sequenced the gene and deduced the amino acid sequence of its product . A sequence homology search revealed that it was same as that of Cmr (or MdfA) . Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter. J Biochem (Tokyo), 1998 Jul, 124(1), 122 - 9 Amino-terminal region of SecA is involved in the function of SecG for protein translocation into Escherichia coli membrane vesicles; Mori H et al.; Protein translocation across the cytoplasmic membrane of Escherichia coli is accomplished by concerted actions of the translocation ATPase SecA and the membrane-embedded SecE/Y/G complex . SecA interacts with preproteins and undergoes ATP-driven cycles of membrane insertion-deinsertion . To address how SecA interacts functionally with other components in the translocation machinery, we characterized a SecA mutant lacking amino-terminal 8 amino acid residues (SecA N-8) . Although the absence of the 8 residues did not grossly affect the interaction of SecA with a preprotein, ATP, or phospholipids, nor did it affect the intrinsic ATPase activity, it gave differential effects on the translocation of different preproteins . It also affected the translocation ATPase activity, the ability of membrane insertion, and the topology inversion of SecG coupled with the membrane insertion-deinsertion of SecA . Most noteworthy, SecA N-8 was pronouncedly defective in the translocation of proton motive force-dependent preproteins, in which SecG might have a role . We propose that the amino-terminal region of SecA is important for the functional interaction with SecG. Mutagenesis, 1998 May, 13(3), 249 - 55 Statistical analysis of lacZ mutant frequency data from MutaMouse mutagenicity assays; Fung KY et al.; Transgenic mouse assays have provided an unprecedented opportunity to study mutagenesis in diverse rodent tissues . In this article data from MutaMouse mutagenicity assays based on the Escherichia coli gene lacZ were analyzed systematically using liver and bone marrow as potential target tissues . Sources of variation, including plates (within packaging reactions), packaging reactions (within animals) and animals, were evaluated for extra-binomial variation . Although hardly any evidence of overdispersion was detected at the plate level, limited evidence of extra-binomial variation was observed at the packaging reaction level . There was, however, much stronger evidence of overdispersion at the animal level . Statistical tests for increasing trend in mutant frequency with increasing dose were also performed at the animal level . A significant increasing trend following exposure to N-nitrosodibenzylamine was detected in liver but not in bone marrow . A logistical model was used to further describe the dose-response relationship observed in N-nitrosodibenzylamine-treated liver tissue. Mol Microbiol, 1998 May, 28(4), 847 - 57 Domain structure and RNA annealing activity of the Escherichia coli regulatory protein StpA; Cusick ME et al.; The Escherichia coli regulatory protein StpA bears striking similarity to the chromatin-associated protein H-NS . These two proteins have many structural, functional and mechanistic parallels . Although H-NS is more abundant in the cell, both proteins act as transcriptional regulators, both bind to curved DNA and both restrain DNA supercoils . However, StpA is better able to promote RNA annealing and trans-splicing in vitro . In this study, phylogenetic analyses and experiments to examine the protease sensitivity of StpA and H-NS suggest a similar structure for the two proteins . Both proteins consist of two structured domains separated by an exposed protease-sensitive linker . The N-terminal (StpA-NterL) and C-terminal (StpA-CterL) domains of StpA, as well as the full-length StpA and H-NS proteins, were cloned, overproduced in E . coli and purified to homogeneity . StpA-CterL, but not StpA-NterL, promotes strand annealing of complementary RNA oligonucleotides and in vitro trans-splicing of a model group I intron . Both StpA and StpA-CterL exhibited stronger RNA-modulating activity than H-NS . Phylogenetic analyses showed that the N-terminal and C-terminal domains can exist autonomously . The phylogenetic and experimental data are compatible with a two-domain model for StpA and H-NS, with independently functioning modules joined by a non-conserved linker, and with the observed RNA-related activities residing entirely within the C-terminal domain. Mol Microbiol, 1998 May, 28(4), 803 - 12 Polypeptide binding of Escherichia coli FtsH (HflB); Akiyama Y et al.; The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease . In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein . A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP . ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH . Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme . Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient . Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated . Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not . Thus, FtsH association with denatured PhoA is uncoupled from proteolysis . Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment . Thus, denatured PhoA binding of FtsH may also occur in vivo. Mol Microbiol, 1998 May, 28(4), 755 - 65 Inducer exclusion by glucose 6-phosphate in Escherichia coli; Hogema BM et al.; The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA(Glc) as a result of transport and phosphorylation of PTS carbohydrates . Dephosphorylation of enzyme IIA(Glc) leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g . lactose, glycerol), and reduced adenylate cyclase activity . In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion . Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-beta-D-thiogalactoside (TMG) . Inhibition was absent in mutants that lacked enzyme IIA(Glc) or were insensitive to inducer exclusion because enzyme IIA(Glc) could not bind to the lactose carrier . Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA(Glc) . In a mutant insensitive to enzyme IIA(Glc)-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells . We discuss an expanded model of enzyme IIA(Glc)-mediated catabolite repression which embodies repression by non-PTS carbon sources. Mol Microbiol, 1998 May, 28(4), 691 - 704 Target specificity of insertion element IS30; Olasz F et al.; The Escherichia coli resident mobile element IS30 has pronounced target specificity . Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence . Insertion sites in phages, plasmids and in the genome of E . coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy . This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion . The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity . Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis . In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target . Having compared the known insertion sites, we suppose that IS30-like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too. Vet Immunol Immunopathol, 1998 Apr 16, 62(3), 197 - 208 Enzymatic amplification and expression of bovine interleukin-1 receptor antagonist cDNA; Kirisawa R et al.; cDNA generated from lipopolysaccharide-stimulated bovine peripheral blood mononuclear cells was used to amplify and clone the bovine interleukin-1 receptor antagonist (IL-1ra) using primers derived from semi-conserved regions between human and mouse IL-1ra sequences . 5' and 3' terminal sequences of bovine IL-1ra were amplified by 5' and 3' rapid amplification of cDNA ends . The deduced amino acid sequence of bovine IL-1ra demonstrated 80%, 78%, 78%, 77% and 76% homology with human, mouse, rat, rabbit and equine sequences, respectively . Recombinant bovine IL-1ra produced in Escherichia coli suppressed the growth inhibitory activity of bovine IL-1beta on A375 cells in a dose-dependent manner, indicating that the present bovine IL-1ra cDNA encodes biologically active proteins. J Pharm Pharmacol, 1998 May, 50(5), 475 - 82 Novel 5,8-diazabenzo{c}phenanthrenes: synthesis and mutagenicity; Upton M et al.; The polycyclic aromatic hydrocarbons have been recognized as carcinogens and mutagens since the early part of this century . More recently their aza and polyaza derivatives have been shown to have the same biological activity . A major source of these compounds is the combustion of fresh or metamorphosed plant materials; this contributes to the environmental burden of, and exposure to, these carcinogens . We report the synthesis and characterization of a series of novel 5,8-diazabenzo{c}phenanthrenes which are isosteric with the known epidermal carcinogen benzo{c}phenanthrene but have not yet been reported as components of soot or diesel particulate matter . The synthesis of the compounds exploits a versatile, double Friedlander reaction between the appropriately substituted 2,2'-diaminobenzophenone and beta-diketones, with yields of purified product ranging from 30-90% . The nucleophilic substitution of these diazabenzophenanthrenes with ethanolamine is also described . This strategy will enable further elaboration of these heterocyclic nuclei at a later date . Mutagenicity testing of these agents was performed using spot tests and in Ames plate-incorporation assays using Escherichia coli WP2 and WP2uvrA as test organisms . The plate-incorporation assays were performed in the presence or absence of metabolic enzymes contained in the S9 liver fraction from Aroclor 1254-induced rats, to investigate whether bioactivation of the diazabenzophenanthrenes contributed to their toxicity . No differences between these two protocols were observed, with neither test showing reversion to prototrophic behaviour . Furthermore, the compounds were not toxic to the test organism . These initial results suggest that these compounds are not mutagenic in the Ames tests employed. Nippon Ronen Igakkai Zasshi, 1998 Apr, 35(4), 303 - 6 {An animal model of aspiration and aspiration pneumonia using lacZ gene expression in lungs by adenoviral vectors}; Teramoto S et al.; To examine the relationship between disturbed upper airway reflexes and aspiration pneumonia, we administered a total volume of 20 microliters of Ad-CMV-lacZ (Ad vector) or 20 microliters of phosphate buffer solution (PBS) intranasal to C57 black mice . In nostrils, the lacZ gene expression was investigated in each mouse with or without anesthesia . Under anesthesia, the lacZ gene expression was detected by Xgal staining in the lungs of every mouse given the Ad vector . However, no gene expression was measured in the lungs of those given the Ad vector without anesthesia . In mice treated with PBS, there was no lacZ gene expression in the nostrils, trachea, or lungs, irrespective of anesthesia . These results suggest that unconsciousness or disturbed upper airway reflexes caused by anesthesia caused aspiration, resulting in an intranasal bolus that can reached the lower airways . This process can be analyzed in mice tracted with adenovirus vectors carrying the E . coli LacZ gene . Mice given Ad-CMV-lacZ transnasally can be used to study aspiration pneumonia in relation to unconsciousness. J Biol Chem, 1998 Jul 3, 273(27), 17251 - 7 Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants; Jansch L et al.; Transport of most nuclear encoded mitochondrial proteins into mitochondria is mediated by heteropolymeric translocases in the membranes of the organelles . The translocase of the outer mitochondrial membrane (TOM) was characterized in fungi, and it was shown that TOM from yeast comprises nine different subunits . This publication is the first report on the preparation of the TOM complex from plant mitochondria . The protein complex from potato was purified by (a) blue native polyacrylamide gel electrophoresis and (b) by immunoaffinity chromatography . On blue native gels, the potato TOM complex runs close to cytochrome c oxidase at 230 kDa and hence only comprises about half of the size of fungal TOM complexes . Analysis of the TOM complex from potato by SDS-polyacrylamide gel electrophoresis allows separation of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa . The 23-kDa protein is identical to the previously characterized potato TOM20 receptor, as shown by in vitro assembly of this protein into the 230-kDa complex, by immunoblotting and by direct protein sequencing . Partial amino acid sequence data of the other subunits allowed us to identify sequence similarity between the 36-kDa protein and fungal TOM40 . Sequence analysis of cDNAs encoding the 7-kDa protein revealed significant sequence homology of this protein to TOM7 from yeast . However, potato TOM7 has a N-terminal extension, which is very rich in basic amino acids . Counterparts to the TOM22 and TOM37 proteins from yeast seem to be absent in the potato TOM complex, whereas an additional low molecular mass subunit occurs . Functional implications of these findings are discussed. J Biol Chem, 1998 Jul 3, 273(27), 17236 - 42 The effects of chaperones and the influence of protein assembly on peroxisomal protein import; Crookes WJ et al.; Peroxisomal proteins are synthesized in the cytoplasm and post-translationally translocated into the organelle . The role of chaperones and protein folding in peroxisomal protein transport is still unclear . Translocation of proteins into mitochondria requires that precursor proteins assume an extended conformation; cytosolic chaperones are thought to help maintain this conformation . In contrast, peroxisomal protein import does not require unfolding of the targeted protein . However, the molecular chaperones Hsp70 and Hsp40 may be important for translocation . We present several lines of evidence that show that plant peroxisomal protein import is enhanced by chaperones . First, peroxisomes isolated from heat-shocked pumpkin seedling tissues exhibited increased protein import relative to control peroxisomes . Second, antibodies raised against wheat germ cytosolic Hsp70 and Escherichia coli Hsp90 inhibited import of the peroxisomal protein isocitrate lyase . To our knowledge, this is the first time that Hsp90 has been directly implicated in a protein transport event . Third, peroxisomal proteins were immunoprecipitated by wheat germ Hsp70 antibodies . We also present results that suggest that the efficiency of peroxisomal protein import is influenced by the structure of the targeted protein; monomeric isocitrate lyase was imported more efficiently than oligomeric isocitrate lyase . Taken together, these data demonstrate that the assembly state of peroxisomal proteins and the chaperones that may mediate those states are both important for efficient peroxisomal protein import. J Biol Chem, 1998 Jul 3, 273(27), 17178 - 85 Arrangement of the multicopy H+-translocating subunit c in the membrane sector of the Escherichia coli F1F0 ATP synthase; Jones PC et al.; The multicopy subunit c of the H+-transporting F1F0 ATP synthase of Escherichia coli is thought to fold across the membrane as a hairpin of two hydrophobic alpha-helices . The conserved Asp61, centered in the second transmembrane helix, is essential for H+ transport . In this study, we have made sequential Cys substitutions across both transmembrane helices and used disulfide cross-link formation to determine the oligomeric arrangement of the c subunits . Cross-link formation between single Cys substitutions in helix 1 provided initial limitations on how the subunits could be arranged . Double Cys substitutions at positions 14/16, 16/18, and 21/23 in helix 1 and 70/72 in helix 2 led to the formation of cross-linked multimers upon oxidation . Double Cys substitutions in helix 1 and helix 2, at residues 14/72, 21/65, and 20/66, respectively, also formed cross-linked multimers . These results indicate that at least 10 and probably 12 subunits c interact in a front-to-back fashion to form a ring-like arrangement in F0 . Helix 1 packs at the interior and helix 2 at the periphery of the ring . The model indicates that the Asp61 carboxylate is centered between the helical faces of adjacent subunit c at the center of a four-helix bundle. J Biol Chem, 1998 Jul 3, 273(27), 17154 - 65 In vitro reconstitution of the recombinant photosystem II light-harvesting complex CP24 and its spectroscopic characterization; Pagano A et al.; The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a . This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures . We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids . Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or beta-carotene . Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules . Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins . A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding . These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions . The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment . Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a. J Biol Chem, 1998 Jul 3, 273(27), 17122 - 7 The fragile-X-related gene FXR1 is a human autoantigen processed during apoptosis; Bolivar J et al.; We describe a new human autoimmune antigen in a patient suffering from scleroderma with high levels of antibodies to nucleolus and cytoplasmic antigens . Using a Chinese hamster ovary cell expression library, we have shown that this antigen corresponds to the autosomal Fragile-X-related gene FXR1 . The deduced amino acid sequence from the hamster cDNA is 97, 98, and 58% homologous to the human, mouse, and Xenopus laevis FXR1 genes, respectively . Expression of the hamster cDNA clone in Escherichia coli and antibody production indicates unequivocally the location of the FXR1 protein in the cytoplasm of hamster cells . Affinity chromatography followed by immunofluorescence microscopy analysis and immunoblots demonstrated the presence of autoimmune IgGs to FXR1 in the scleroderma patient . Immunolabeling studies in Jurkat cells, induced to apoptosis by anti-Fas/APO1 serum, indicated that the FXR1 antigens were clearly displaced from their original cytoplasmic location to several punctuated foci, resembling the bleb-like membranous structures characteristic of cells at certain stages of apoptosis . This phenomenon could be part of a putative mechanism in which the FXR1 protein is presented as a target for the autoimmune response in humans. J Biol Chem, 1998 Jul 3, 273(27), 17109 - 14 The amino-terminal domain of human STAT4 . Overproduction, purification, and biophysical characterization; Baden HA et al.; The multifunctional signal transducer and activator of transcription (STAT) proteins relay signals from the cell membrane to the nucleus in response to cytokines and growth factors . STAT4 becomes activated when cells are treated with interleukin-12, a key cytokine regulator of cell-mediated immunity . Upon activation, dimers of STAT4 bind cooperatively to tandem interferon-gamma activation sequences (GAS elements) near the interferon-gamma gene and stimulate its transcription . The amino-terminal domain of STAT4 (STAT4(1-124)) is required for cooperative binding interactions between STAT4 dimers and activation of interferon-gamma transcription in response to interleukin-12 . We have overproduced this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and purified it to homogeneity for structural studies . The circular dichroism spectrum of hSTAT4(1-124) indicates that it has a well ordered conformation in solution . The translational diffusion constant of hSTAT4(1-124) was determined by nuclear magnetic resonance methods and found to be consistent with that of a dimer . The rotational correlation time (tauc) of hSTAT4(1-124) was estimated from 15N relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric protein . These results, together with the number of signals observed in the two-dimensional 1H-15N heteronuclear single quantum coherence spectrum of uniformly 15N-labeled protein, indicate that hSTAT4(1-124) forms a stable, symmetric homodimer in solution . Cooperativity in native STAT4 probably results from a similar or identical interaction between the amino-terminal domains of adjacent dimers bound to DNA. J Biol Chem, 1998 Jul 3, 273(27), 17036 - 49 Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase; Bridges A et al.; A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O . B., Vakser, I . A., Loew, G . H., Bridges, A., and Waskell, L . (1997) Protein Eng . 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b5 . Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis . These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b5 and support substrate oxidation . Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b5 . All of the mutants except K433A are located in either the C or C* helices or their termini . In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase . These studies indicate that the binding sites for cytochrome b5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical. J Biol Chem, 1998 Jul 3, 273(27), 17025 - 35 A B-cell-specific DNA recombination complex; Borggrefe T et al.; We have purified and biochemically characterized a multiprotein complex designated SWAP . In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions . We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70 . The first three are proteins known to be present in most cells . B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences . SWAP-70 is a novel protein of 70 kDa . Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli . SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines . SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase . These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components. J Biol Chem, 1998 Jul 3, 273(27), 16860 - 4 Conversion of temperature-sensitive to -resistant gene expression due to mutations in the promoter region of the melibiose operon in Escherichia coli; Tamai E et al.; The melibiose utilization system of Escherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 degreesC . The reason for this temperature sensitivity was thought to be that the melibiose transporter (MelB) of W3133 cells was temperature-sensitive . A mutant W3133-2 has been isolated as a temperature-resistant strain that can utilize melibiose between 37 and 42 degreesC . However, we found that the melibiose transporter of the W3133-2 was still temperature-sensitive . Half-life activities of the melibiose transporter at 37 degreesC (or 40 degreesC) in both E . coli W3133 and W3133-2 were exactly the same . Furthermore, we found that the nucleotide sequence of coding region of the melB structural gene (the second gene of the melibiose operon) of W3133-2 was exactly the same as that of W3133 . Activity of alpha-galactosidase (product of the first gene, melA, of the melibiose operon) of W3133 cells grown at 40 degreesC was very low, although that of W3133-2 cells grown at 40 degreesC was high . These observations suggested that expression of the melibiose operon in W3133 is also temperature-sensitive . In fact, we found that the expression in W3133 cells was temperature-sensitive, while that in W3133-2 cells was temperature-resistant, by analyzing mRNA levels using the Northern blot method . Furthermore, we identified mutations in the promoter region of the melibiose operon of W3133-2 that resulted in the elongation of an 18 nucleotide inverted repeat sequence to a 28-nucleotide repeat sequence present immediately upstream of the -35 region . This may stabilize a possible stem structure due to the inverted repeat at 37-42 degreesC. J Biol Chem, 1998 Jul 3, 273(27), 16816 - 23 Cathepsin Z, a novel human cysteine proteinase with a short propeptide domain and a unique chromosomal location; Santamaria I et al.; We have identified and characterized a novel human cysteine proteinase of the papain family . A full-length cDNA for this enzyme was cloned from a human brain cDNA library . Nucleotide sequence analysis revealed that the isolated cDNA codes for a polypeptide of 303 amino acids, tentatively called cathepsin Z, that exhibits structural features characteristic of cysteine proteinases . Fluorescent in situ hybridization experiments revealed that the human cathepsin Z gene maps to chromosome 20q13, a location that differs from all cysteine proteinase genes mapped to date . The cDNA encoding cathepsin Z was expressed in Escherichia coli as a fusion protein with glutathione S-transferase, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, used as a substrate for cysteine proteinases . Northern blot analysis demonstrated that cathepsin Z is widely expressed in human tissues, suggesting that this enzyme could be involved in the normal intracellular protein degradation taking place in all cell types . Cathepsin Z is also ubiquitously distributed in cancer cell lines and in primary tumors from different sources, suggesting that this enzyme may participate in tumor progression as reported for other cathepsins . Finally, on the basis of a series of distinctive structural features, including diverse peptide insertions and an unusual short propeptide, together with its unique chromosomal location among cysteine proteinases, we propose that cathepsin Z may be the first representative of a novel subfamily of this class of proteolytic enzymes. J Biol Chem, 1998 Jul 3, 273(27), 16678 - 85 A novel metal-activated pyridoxal enzyme with a unique primary structure, low specificity D-threonine aldolase from Arthrobacter sp . Strain DK-38 . Molecular cloning and cofactor characterization; Liu JQ et al.; The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/D-allo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp . strain DK-38 . The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues . The enzyme was overproduced in recombinant Escherichia coli cells and purified to homogeneity by ammonium sulfate fractionation and three-column chromatography steps . The recombinant aldolase was identified as a pyridoxal enzyme with the capacity of binding 1 mol of pyridoxal 5'-phosphate per mol of subunit, and Lys59 of the enzyme was determined to be the cofactor binding site by chemical modification with NaBH4 . In addition, Mn2+ ion was demonstrated to be an activator of the enzyme, although the purified enzyme contained no detectable metal ions . Equilibrium dialysis and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn2+ ion per mol of subunit . Remarkably, the predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5'-phosphate-dependent enzymes, indicating that low specificity D-threonine aldolase is a new pyridoxal enzyme with a unique primary structure . Taken together, low specificity D-threonine aldolase from Arthrobacter sp . strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme. J Bacteriol, 1998 Jul, 180(13), 3486 - 90 Aberrant cell division and random FtsZ ring positioning in Escherichia coli cpxA* mutants; Pogliano J et al.; In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division . However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis . It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division. J Bacteriol, 1998 Jul, 180(13), 3462 - 6 Does disparate occurrence of autoregulatory programmed frameshifting in decoding the release factor 2 gene reflect an ancient origin with loss in independent lineages? Persson BC, Atkins JF. In Escherichia coli an autoregulatory mechanism of programmed ribosomal frameshifting governs the level of polypeptide chain release factor 2 . From an analysis of 20 sequences of genes encoding release factor 2, we infer that this frameshift mechanism was present in a common ancestor of a large group of bacteria and has subsequently been lost in three independent lineages. J Bacteriol, 1998 Jul, 180(13), 3448 - 52 The A1A0 ATPase from Methanosarcina mazei: cloning of the 5' end of the aha operon encoding the membrane domain and expression of the proteolipid in a membrane-bound form in Escherichia coli; Ruppert C et al.; Three additional ATPase genes, clustered in the order ahaH, ahaI, and ahaK, were found upstream of the previously characterized genes ahaECFABDG coding for the archaeal A1A0 ATPase from Methanosarcina mazei . ahaH, the first gene in the cluster, is preceded by a conserved promoter sequence . Northern blot analysis revealed that the clusters ahaHIK and ahaECFABDG are transcribed as one message . AhaH is a hydrophilic polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated ATPase genes in Methanobacterium thermoautotrophicum and Methanococcus jannaschii . AhaI has a two-domain structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane alpha helices . It is similar to the 100-kDa polypeptide of V1V0 ATPases and is therefore suggested to participate in proton transport . AhaK is a hydrophobic polypeptide with two predicted transmembrane alpha helices and, on the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide which is essential for proton translocation . However, it is only one-half and one-third the size of the proteolipids from M . thermoautotrophicum and M . jannaschii, respectively . ahaK is expressed in Escherichia coli, and it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea and bacteria . This is the first report on the expression and incorporation into E . coli lipids of a membrane integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the membrane domain of the methanoarchaeal ATPase. J Bacteriol, 1998 Jul, 180(13), 3441 - 7 Membrane-bound lytic endotransglycosylase in Escherichia coli; Kraft AR et al.; The gene for a novel endotype membrane-bound lytic transglycosylase, emtA, was mapped at 26.7 min of the E . coli chromosome . EmtA is a lipoprotein with an apparent molecular mass of 22kDa . Overexpression of the emtA gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment . The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme . The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism . EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier. J Bacteriol, 1998 Jul, 180(13), 3388 - 92 Identification and characterization of two quiescent porin genes, nmpC and ompN, in Escherichia coli BE; Prilipov A et al.; The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far . Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes . The two genes were cloned and sequenced . One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification . The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E . coli K-12 . The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E . coli BE. J Bacteriol, 1998 Jul, 180(13), 3345 - 52 Induction of the SOS response increases the efficiency of global nucleotide excision repair of cyclobutane pyrimidine dimers, but not 6-4 photoproducts, in UV-irradiated Escherichia coli; Crowley DJ et al.; Nucleotide excision repair (NER) is responsible for the removal of a variety of lesions from damaged DNA and proceeds through two subpathways, global repair and transcription-coupled repair . In Escherichia coli, both subpathways require UvrA and UvrB, which are induced following DNA damage as part of the SOS response . We found that elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampin reduced the efficiency of global repair of the major UV-induced lesion, the cyclobutane pyrimidine dimer (CPD), but had no effect on the global repair of 6-4 photoproducts . Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampin . Transcription-coupled repair of CPDs occurred in the absence of SOS induction but was undetectable when the response was expressed constitutively . These results suggest that damage-inducible synthesis of UvrA and UvrB is necessary for efficient repair of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts . We compare our findings with recent data from eukaryotic systems and suggest that damage-inducible stress responses are generally critical for efficient global repair of certain types of genomic damage. Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 234 - 41 Characterization of human mitochondrial RNase P: novel aspects in tRNA processing; Rossmanith W et al.; Human mitochondrial RNase P does not distinguish itself from other RNase P enzymes by most of its basic properties . 5' phosphates on tRNA products, strict dependence on a divalent cation, independence of ATP or other cofactors, and sensitivity to puromycin are generally characteristic for RNase P . Slow sedimentation of human mitochondrial RNase P in glycerol gradients suggests a molecular weight considerably lower than that of bacterial or nuclear RNase P . In contrast to fungi, all putative components of mammalian mitochondrial RNase P are encoded by the nucleus . Intriguingly, no indication of the involvement of a trans-acting RNA was found in mammalian mitochondrial tRNA processing . Mitochondrial RNase P is resistant to rigorous treatments with nucleases and exhibits a protein-like density in Cs2SO4 gradients . Moreover, an analysis of copurifying RNAs revealed no putative RNase P RNA candidates . These data suggest that mammalian mitochondrial RNase P, unlike its nuclear counterpart or its bacterial relatives, is not a ribonucleoprotein but a protein enzyme . Protein Expr Purif . 1998 Jun;13(1):iv. Papers to Appear in Forthcoming Issues; Exploring the folding pathways of annexin I et al.; Section de Biophysique des Proteines et des Membranes and URA CNRS 2096, Gif sur Yvette Cedex, CEA Saclay, 91191, FranceIn the context of exploring the relationship between sequence and folding pathways, the multi-domain proteins of the annexin family constitute very attractive models . They are constituted of four approximately 70-residue domains, named D1 to D4, with identical topologies but only limited sequence homology of approximately 30% . The domains are organized in a pseudochiral circular arrangement . Here, we report on the folding propensity of the D1 domain of annexin I obtained from overexpression in Escherichia coli . Unlike the D2 domain, which is only partially folded, the isolated D1 domain exhibits autonomous refolding in pure aqueous solution . Similarly, the D3 domain and D2-D3 module were obtained from expression in E . coli but were found to be largely unfolded . No conclusion could be drawn for the D4 domain because it was not possible to extract it from the bacterial inclusion bodies . The data allow us to propose a plausible scenario for the annexin I folding . This working model states that firstly the D1 domain folds, and the D2 and D3 domains remain partly unfolded, facilitating the docking of the D4 domain to the D1 domain . In a second step, the D1 and D4 domains dock, and D4 may fold if already not folded . The final step starts with the stabilization of the D1-D4 module . This stabilization is crucial for allowing the non-native local interactions inside the still partially unfolded D2 domain to switch to the native long-range interactions involving D4 . This switch allows the complete folding of D2 and D3 . The model proposes a sequential and hierarchical process for the folding of annexin I and emphasizes the role of both native framework and non-native structures in the process . J Mol Biol, 1998 Jun 26, 279(5), 1163 - 75 Exploring the folding pathways of annexin I, a multidomain protein . I . non-native structures stabilize the partially folded state of the isolated domain 2 of annexin I; Cordier-Ochsenbein F et al.; Proteins of the annexin family constitute very attractive models because of their four approximately 70 residue domains, D1 to D4, exhibiting an identical topology comprising five helix segments with only a limited sequence homology of approximately 30% . We focus on the isolated D2 domain, which is only partially folded . A detailed analysis of this equilibrium partially folded state in aqueous solution and micellar solution using 15N-1H multidimensional NMR is presented . Comparison of the residual structure of the entire domain with that of shorter fragments indicates the presence of long-range transient hydrophobic interactions that slightly stabilize the secondary structure elements . The unfolded domain tends to behave as a four-helix, rather than as a five-helix domain . The ensemble of residual structures comprises: (i) a set of native structures consisting of three regions with large helix populations, in rather sharp correspondence with A, B and E helices, and a small helix population in the second part of the C helix; (ii) a set of non-native local structures corresponding to turn-like structures stabilized by several side-chain to side-chain interactions and helix-disruptive side-chains to backbone interactions . Remarkably, residues involved in these local non-native interactions are also involved, in the native structure, in structurally important non-local interactions . During the folding process of annexin I, the local non-native interactions have to switch to native long-range interactions . This structural switch reveals the existence of a sequence-encoded regulation of the folding pathways and kinetics, and emphasizes the key role of the non-native local structures in this regulation . J Mol Biol, 1998 Jun 26, 279(5), 1101 - 10 Expansion of CTG repeats from human disease genes is dependent upon replication mechanisms in Escherichia coli: the effect of long patch mismatch repair revisited; Schumacher S et al.; Many human hereditary disease genes have been recently associated with the expansion of CTG/GAC repeats . We have used a plasmid-based assay in Escherichia coli to investigate the instability of a (CTG/GAC) insert containing 64 repeats . Using this assay, expansions were biochemically detected and subsequently quantified . We show that the occurence of expansions within these trinucleotide repeats is dependent upon replicative mechanisms . Expansions of up to 30 repeats and deletions of almost all possible sizes occured regardless of the orientation of the insert relative to the replication origin . In contradiction to a previous report, the mismatch repair pathway was found to strongly stabilize these repeat stretches . J Mol Biol, 1998 Jun 26, 279(5), 1075 - 83 Cooperative interaction between the DNA-binding domains of PU.1 and IRF4; Yee AA et al.; The two lymphoid-specific transcription factors PU.1 and IRF4 form a cooperative ternary complex at immunoglobulin enhancer elements such as the lambdaB and kappaE3' sites . We report here that the synergy of this interaction can be reconstituted in part with the DNA-binding domains of the two proteins . The minimal DNA binding-domain of IRF4 was mapped to residues 20 to 137, corresponding to the conserved DNA-binding region of other interferon regulatory factors (IRFs) . This domain can bind weakly to a synthetic murine lambdaB element, while IRF4 constructs that contain residues 1 to 19 require the presence of PU.1 for DNA-binding at similar concentrations.Fluorescence polarization of fluorescein-labelled DNA was used to show that the presence of residues 1 to 19 decreases the binding affinity of IRF4 N-terminal constructs from two- to fivefold . However, all constructs bound better to the lambdaB element in the presence of the DNA-binding domain of PU.1 . This cooperative interaction was not dependent on phosphorylation of the PEST domain of PU.1, but was dependent on the proper spacing of the binding sites for PU.1 and IRF4 . These data suggest that at least part of the cooperative interaction between full-length PU.1 and IRF4 involves the DNA-binding domains of the two proteins . NMR spectroscopy of 15N-labelled PU.1 and IRF4 constructs indicates that the PEST domain of PU.1 and residues 1 to 19 of IRF4 may be unstructured in the isolated proteins . J Mol Biol, 1998 Jun 26, 279(5), 1061 - 74 Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes; Coburn GA et al.; Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli . We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase . As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro . However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated . Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II . Moreover, both ATP and a non-hydrolyzable analog, ATPgammaS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required . Alternatively, the rpsT mRNA can be degraded in vitro by a second 3'-decay pathway which is dependent on PAP I, PNPase and ATP alone . Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3' termini . Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined . J Mol Biol, 1998 Jun 26, 279(5), 1045 - 51 Tyrosine phosphorylation in Escherichia coli; Freestone P et al.; The phosphorylation on tyrosine of a protein in Escherichia coli both in vivo and in vitro was revealed by recognition by anti-phosphotyrosine antibodies, labelling with {gamma-32P}ATP, and phosphoamino acid analysis . This protein, which we name TypA, is the product of the o591 reading frame as revealed by N-terminal sequencing and antibody cross-reactivity . Inactivation of typA altered the patterns of protein synthesis during both exponential growth and carbon starvation . These alterations included the disappearance of an acidic isoform of both the universal stress protein UspA and carbon starvation protein Csp15, and increased synthesis of the histone-like protein H-NS . The sequence of TypA from strain K-12 differs from that of an enteropathogenic strain in six amino acid residues and the protein is three residues shorter . We propose that TypA interacts with global regulatory networks and that its phosphorylation may be relevant to pathogenesis . J Mol Biol, 1998 Jun 19, 279(4), 1001 - 11 Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator; Yang SQ et al.; Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design . An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA) . The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site . Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop . The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds . Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence . Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70 . The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT) . A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin . Panning against rat trypsin resulted in enrichment but no consensus sequence . Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70 . Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT . Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin . The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold . J Mol Biol, 1998 Jun 19, 279(4), 945 - 57 Ecotin: a serine protease inhibitor with two distinct and interacting binding sites; Yang SQ et al.; The interaction between ecotin and target proteases with trypsin-like specificity has been systematically dissected to understand the structural basis of ecotin's broad inhibitory specificity and the role of the secondary binding site . Site-directed and region-specific mutagenesis were preformed at ecotin's primary site P1 residue (84), the C-terminal dimer interface (133 to 142), and two surface loops of the secondary binding site (67 to 70, 108 to 113) . Substitutions at the P1 position resulted in less than fivefold difference in the potency of ecotin binding to rat trypsin, suggesting that the extended binding site is important in binding . A ten amino acid C-terminal truncation variant showed threefold weaker self-association but remained a dimer . The interactions of the secondary binding site of ecotin with bovine trypsin, rat trypsin and human urokinase-type plasminogen activator (uPA) were investigated with alanine substitutions in ecotin at Trp67, Gly68, Tyr69, Asp70, Arg108, Asn110, Lys112 and Leu113, which formed contacts between the inhibitor and protease . By combining these mutations at the secondary binding site with mutations in the primary binding site the molecular recognition between ecotin and its target serine proteases was probed . The contrast in the Ki values of the various ecotin variants towards bovine trypsin, rat trypsin and human uPA established the role of ecotin's secondary binding site in recognizing these homologous serine proteases . Ecotin binds to proteases with a chymotrypsin fold through a combination of primary and secondary site surface loops and is amenable to redesign of its potency and specificity for this class of enzymes . J Mol Biol, 1998 Jun 19, 279(4), 833 - 40 Catapult mechanism renders the chaperone action of Hsp70 unidirectional; Gisler SM et al.; Molecular chaperones of the Hsp70 type promote the folding and membrane translocation of proteins . The interaction of Hsp70s with polypeptides is linked to ATP binding and hydrolysis . We formed complexes of seven different fluorescence-labeled peptides with DnaK, the Hsp70 homolog of Escherichia coli, and determined the rate of peptide release under two different sets of conditions . (1) Upon addition of ATP to nucleotide-free peptide.DnaK complexes, all tested peptides were released with similar rate constants (2.2 s-1 to 6.7 s-1) . (2) In the binding equilibrium of peptide and ATP-liganded DnaK, the dissociation followed one or two-step reactions, depending on the amino acid sequence of the peptide . For the monophasic reactions, the dissociation rate constants diverged by four orders of magnitude from 0.0004 s-1 to 5.7 s-1; for the biphasic reactions, the rate constants of the second, slower isomerization step were in the range from 0.3 s-1 to 0.0005 s-1 . The release of the different peptides in case (1) is 1.4 to 14,000 times faster than in case (2) . Apparently, binding of ATP induces a transient state of the chaperone which ejects target peptides before the final state of ATP-liganded DnaK is reached . This "catapult" mechanism provides the chaperone cycle with a mode of peptide release that does not correspond with the reverse of peptide binding . By allowing the conformation of the outgoing polypeptide to differ from that of the incoming polypeptide, a futile cycle with respect to conformational work exerted on the target protein is obviated . J Mol Biol, 1998 Jun 19, 279(4), 823 - 32 Analyzing the functional organization of a novel restriction modification system, the BcgI system; Kong H; BcgI is a novel, multi-subunit, restriction-modification (R-M) system that differs from all the other types of R-M system in its genetic and functional organization . The holoenzyme contains two different subunits, BcgI A and BcgI B . Both are required for endonuclease and methyltransferase activities . Here, we show that the endonuclease activity is mediated by the N-terminal portion of the A subunit . We made this assignment by mutational analysis . The analytic strategy involved three steps . First, the methyltransferase activity was inactivated by site-directed mutagenesis of a conserved methyltransferase motif also found in the A subunit . One of the R+M- mutants could not methylate DNA but was still able to cleave it, therefore expression of this mutant gene was lethal to the host . This lethal phenotype allowed the selective isolation of cleavage-deficient (R-) mutations in a second round of random mutagenesis in this mutant background . The R- mutations were all located in the N-terminal portion of the A subunit . There are five potential endonuclease motifs within this region . Conserved acidic residues in each of these motifs were substituted with alanine by site-directed mutagenesis of the wild-type A gene . The results identified one motif, P52E53-(X)12-E66D67K68, as the probable endonuclease active-site . Further support for this assignment was obtained by another round of site-directed mutagenesis directed to residues surrounding this motif . The results showed that DNA cleavage activity was mediated by the predicted, conserved residues, and not any of the surrounding non-conserved residues . One mutant protein, BcgI-E53A, with a single amino acid substitution decreased the DNA cleavage activity at least 700-fold . Our present model for the functional organization of BcgI locates both endonuclease and methyltransferase domains in the A subunit, with the target recognition domain located in the B subunit . J Mol Biol, 1998 Jun 19, 279(4), 753 - 60 A superrepressor mutant of the arginine repressor with a correctly predicted alteration of ligand binding specificity; Niersbach H et al.; Arginine biosynthesis in Escherichia coli is negatively regulated by the hexameric repressor protein ArgR and the corepressor L-arginine . L-Arginine binds to ArgR in the C-terminal domain of the repressor . Binding to operator DNA occurs in the N-terminal domain . The molecular structures of both domains have recently been elucidated . The known stereochemistry of the arginine binding pocket was used for the rational design of a mutant ArgR with altered ligand specificity . Our prediction was that a replacement of Asp128 by asparagine would preferentially lead to the binding of L-citrulline, rather than L-arginine . The D128N mutant was constructed and was shown to fulfill our expectation by several experimental approaches . By isothermal titration calorimetry it was found to bind L-citrulline much more strongly than L-arginine, in contrast to wild-type ArgR . Exchange between the mutant trimers of the hexamer was inhibited by L-citrulline, as it is by L-arginine in the wild-type . The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein . Demonstration of a corepressor action was, however, precluded by the superrepressor effect of the D128N mutation by itself . The mutant protein, in the absence of L-citrulline or L-arginine is as strong a repressor as the wild-type protein in the presence of L-arginine . We discuss two possible mechanisms, in terms of the known domain structures that could explain our observations . J Mol Biol, 1998 Jun 19, 279(4), 727 - 36 tRNA imbalance promotes -1 frameshifting via near-cognate decoding; O'Connor M; tRNAGly1 is the Escherichia coli glycine tRNA specific for GGG codons . A genetic selection for multicopy suppressors of a frameshift mutation has shown that increased levels of wild-type tRNAGly1 causes -1 frameshifting . Analysis of the suppression spectrum of this multicopy suppressor and peptide sequencing of the suppressed protein product showed that it promoted GG doublet decoding at the near-cognate GGA codons . It is proposed that increasing the concentration of the GGG-specific tRNAGly1 relative to the cognate GGA-decoding tRNAGly2 allows the near-cognate tRNA to read GGA codons . Near-cognate decoding of GGA codons by tRNAGly1 can occur by a two-out-of-three reading mechanism, in which only the first two bases of the GGA codon are paired with the anticodon, thus permitting doublet translocations . In mycoplasmas, a single tRNA typically decodes all four triplets of a codon family and introduction of a feature of the Mypoplasma mycoides tRNAGly responsible for non-discriminate decoding, a C at position 32, into the anticodon E . coli tRNAGly1, enhanced the efficiency of doublet decoding . J Mol Biol, 1998 Jun 19, 279(4), 713 - 26 Effects of reaction conditions on RNA secondary structure and on the helicase activity of Escherichia coli transcription termination factor Rho; Walstrom KM et al.; The ATPase and helicase activities of the Escherichia coli transcription termination protein rho have been studied under a variety of reaction conditions that alter its transcription termination activity . These conditions include KCl, KOAc, or KGlu concentrations from 50 to 150 mM and Mg(OAc)2 concentrations from 1 to 5 mM (in the presence of 1 mM ATP) . In higher KCl or higher Mg(OAc)2 concentrations we found that the translocation of rho hexamers along RNA was slower and less processive than the same process measured at 50 mM monovalent salt concentrations and 1 mM Mg(OAc)2 . The ATPase activity of rho was also decreased under reaction conditions that slowed translocation . RNA melting experiments showed that the decreased ATPase activity of rho and the slower helicase activity at increased KCl or Mg(OAc)2 concentrations are accompanied by a concomitant increase in the secondary structure of the RNA portion of the helicase substate . In contrast, the ATPase activity of rho in the presence of poly(rC), a synthetic RNA that does not form salt-concentration-dependent secondary structure, was shown to be the same in each of the three monovalent salts . Thus, the salts do not directly affect the structure or conformation of the rho protein or the binding of rho to single-stranded RNA . However, the translocation of rho along RNA was more processive in 150 mM KOAc or KGlu than in 150 mM KCl, while the RNA secondary structure was the same in all three monovalent salts . Therefore, the monovalent salt present in the reaction may directly affect rho-RNA interactions when the RNA substrate can form secondary structure . Helicase experiments with an RNA molecule that does not contain a rho loading-site showed that rho translocates less processively along this potential helicase substrate . These results suggest that the helicase activity of rho may be significantly regulated by RNA secondary structure . In addition, one of the mechanisms to concentrate the activity of rho on transcripts containing unstructured rho loading sites may be that rho translocation along such molecules is more processive than it is along more structured RNA molecules in the cell . J Mol Biol, 1998 Jun 5, 279(2), 473 - 81 Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues; Skirgaila R et al.; According to the crystal structure of Cfr10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site . Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function . Structural comparison indicates that the D134 residue of Cfr10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX19EAK and 73PDX15DIK of EcoRI and EcoRV, respectively . However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX55KX13E corresponds to the active site of Cfr10I, but differs from canonical active site motifs of EcoRI and EcoRV . According to the crystal structure of Cfr10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between EcoRI and EcoRV . Site-directed mutagenesis experiments of Cfr10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 A away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr10I . In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains . The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX55KX13E of Cfr10I was changed to a canonical motif 133PDX53EVK, which is similar to that of EcoRI and EcoRV . Interestingly, the double mutant S188E/E204S of Cfr10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a gamma DNA cleavage assay . Thus, structure guided redesign of the catalytic/metal binding site of Cfr10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites. J Mol Biol, 1998 Jun 5, 279(2), 439 - 47 Conformational changes of the Tet repressor induced by tetracycline trapping; Orth P et al.; The X-ray crystal structure analysis of inducer-free Tet repressor, TetR, at 2.4 A resolution identifies one of two openings of the tunnel-like binding site as the entrance for the inducer tetracycline-Mg2+, {Mg Tc}+ . Recognition and binding of the inducer unleashes conformational changes leading to the induced state of TetR . In the first step, the C-terminal turn of alpha-helix 6 unwinds, thereby altering the orientation of alpha-helix 4 . This different orientation of alpha-helix 4 is stabilized by a series of hydrogen bonds mediated through a chain of eight water molecules . The alpha-helix 4 connects the DNA-binding domain (alpha-helices 1 to 3) to the rigid TetR core, and thus regulates gene expression through its respective orientations. J Mol Biol, 1998 Jun 5, 279(2), 423 - 38 Analysis of striated fiber formation by recombinant SF-assemblin in vitro; Lechtreck KF; The basal bodies of green flagellates are often connected to striated microtubule-associated fibers (SMAFs), which are highly ordered bundles of 2 nm filaments . SF-assemblin (33 kDa) is the principal structural subunit of the SMAFs and consists of a non-helical head domain of approximately 32 residues and an alpha-helical rod domain that shows a pronounced coiled-coil forming ability . To investigate the functional role of the head domain we expressed N-terminally truncated molecules using a cDNA coding for SF-assemblin from Chlamydomonas reinhardtii . Recombinant wild-type SF-assemblin or molecules with an N-terminal deletion of ten residues formed striated fibers with an axial repeat of 28 nm . N-terminal truncations of 19 and 29 residues yielded assembly-incompetent molecules, revealing that the head domain is necessary for the constitution of striated fibers . Further, molecules with an internal deletion of 24 residues or with duplicated segments corresponding to insertions of 29 and 58 residues were constructed . The resulting fibers had altered cross-striation patterns and axial repeats . The observed shifts in the axial repeat corresponded well to the number of inserted or deleted residues, indicating a linear coherence between molecule length and axial repeat . The heptad pattern of the rod domain of SF-assemblin is regularly interrupted by skip residues . The removal of one or two skip residues had no significant effect on the ultrastructure of the striated fibers . Substitution of skip no . 2 with alanine resulted in a modified, asymmetric cross-striation pattern, indicating a polar architecture of the striated fibers . In summary, various mutations of SF-assemblin effected the solubility of the molecules, and the axial repeat, cross-striation pattern, or overall appearance of the fibers . Thus, analysis of SF-assemblin may represent a valuable system to study the interactions involved in the polymerization of fibrous coiled-coil proteins . A model of the SMAFs based on staggered protofilaments consisting of overlapping 36 nm subunits is presented. J Mol Biol, 1998 Jun 5, 279(2), 403 - 21 Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution; Dube P et al.; Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution . A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites . In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle . On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits . In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle . By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E . coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively . A similar comparison of the secondary structures of mammalian 28 S rRNA and E . coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups . The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area . The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit . The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit. J Mol Biol, 1998 Jun 5, 279(2), 331 - 45 RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters; Bartlett MS et al.; Mutations in Escherichia coli rpoB or rpoC, selected for the ability to confer prototrophy on relA spoT strains, were found to affect transcription from rrn P1 promoters . Two mutant strains (beta RH454 and beta' delta 215-220) reduced transcription of rrn P1 core promoter-lacZ fusions but not of control promoter-lacZ fusions . Purified mutant RNAPs formed complexes with rrn P1 promoters that were much less stable than those formed by wild-type RNAP and required high concentrations of the initiating NTP for efficient rrn P1 transcription . The instability of the rrn P1 core promoter complexes with the mutant RNAPs and their altered regulatory properties support a recently proposed model for the control of rRNA transcription by changing concentrations of the initiating NTPs . We further suggest that destabilization of promoter complexes by the mutant RNAPs mimics effects of ppGpp, decreasing or increasing transcription depending on the kinetic properties of the specific promoter. J Mol Biol, 1998 Jun 12, 279(3), 565 - 76 Combinatorial selection of a small RNA that induces amplification of IncFII plasmids in Escherichia coli; Ferber MJ et al.; Cellular RNAs play fundamental roles as genetic messages, structural components and, in some cases, as catalytic agents . The ability to create vast combinatorial libraries of random RNA sequences has previously been exploited in vitro to identify RNA aptamers with desirable binding specificities, and to isolate RNAs with novel catalytic properties . Despite the advantages of in vitro selections from RNA libraries, there is no way to predict if the identified RNAs can function in living cells . We are therefore exploring random RNA expression libraries in Escherichia coli to search for small RNAs with novel functions . Here we describe selections that identified a small RNA (approximately 260 nucleotides) capable of altering the copy-number control circuitry of IncFII plasmids . The novel RNA appears to function by annealing to a region of the mRNA encoding the plasmid replicator protein . The resulting RNA-RNA hybrid permits translation of the replicator protein, but blocks base-pairing with a natural negative regulatory RNA . Implications of this in vivo selection strategy are discussed. J Mol Biol, 1998 Jun 12, 279(3), 651 - 64 Multiple open forms of ribose-binding protein trace the path of its conformational change; Bjorkman AJ et al.; Conformational changes are necessary for the function of bacterial periplasmic receptors in chemotaxis and transport . Such changes allow entry and exit of ligand, and enable the correct interaction of the ligand-bound proteins with the membrane components of each system . Three open, ligand-free forms of the Escherichia coli ribose-binding protein were observed here by X-ray crystallographic studies . They are opened by 43 degrees, 50 degrees and 64 degrees with respect to the ligand-bound protein reported previously . The three open forms are not distinct, but show a clear relationship to each other . All are the product of a similar opening motion, and are stabilized by a new, almost identical packing interface between the domains . The changes are generated by similar bond rotations, although some differences in the three hinge segments are needed to accommodate the various structural scenarios . Some local repacking also occurs as interdomain contacts are lost . The least open (43 degrees) form is probably the dominant one in solution under normal conditions, although a mixture of species seems likely . The open and closed forms have distinct surfaces in the regions known to be important in chemotaxis and transport, which will differentiate their interactions with the membrane components . It seems certain that the conformational path that links the forms described here is that followed during ligand retrieval, and in ligand release into the membrane-bound permease system. J Mol Biol, 1998 Jun 12, 279(3), 589 - 603 Epitopes fused to F-pilin are incorporated into functional recombinant pili; Rondot S et al.; In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken . Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA . Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin . All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin . The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1 . Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease . Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function . The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope . Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody . A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy . While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success . Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly. J Mol Biol, 1998 Jun 12, 279(3), 513 - 27 Recognition of core-type DNA sites by lambda integrase; Tirumalai RS et al.; Escherichia coli phage lambda integrase (Int) is a 40 kilodalton, 356 amino acid residue protein, which belongs to the lambda Int family of site-specific recombinases . The amino-terminal domain (residues 1 to 64) of Int binds to "arm-type" DNA sites, distant from the sites of DNA cleavage . The carboxy-terminal fragment, termed C65 (residues 65 to 356), binds "core-type" DNA sites and catalyzes cleavage and ligation at these sites . It has been further divided into two smaller domains, encompassing residues 65 to 169 and 170 to 356, respectively . The latter has been characterized and its crystal structure has been determined . Although this domain catalyzes the cleavage and rejoining of DNA strands it, unexpectedly, does not form electrophorectically stable complexes with core-type DNA . Here we have investigated the critical features of lambda Int binding to core-type DNA sites; especially, the role of the central 65 to 169 domain . To eliminate the complexities arising from lambda Int's heterobivalency we studied Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core-type sites . Zero-length UV crosslinking was used to show that Ala125 and Ala126 make close contact with bases in the core-type DNA . Modification by pyridoxal 5'-phosphate was used to identify Lys103 at the protein-DNA interface . Since both of the identified loci are in the central domain, it was cloned and purified and found to bind to core-type DNA autonomously and specifically . The synergistic roles of the catalytic and the central, or core-binding (CB), domains in the interaction with core-type DNA are discussed for (Int and related DNA recombinases. Acta Paediatr, 1998 May, 87(5), 491 - 3 Adhesion inhibitory activity of beta-lactoglobulin isolated from infant formulae; Ouwehand AC et al.; Beta-lactoglobulin was isolated from infant formulae that were ultra high temperature (UHT) -treated, sterilized or spray-dried . The effect of the isolated beta-lactoglobulin on SfaII-fimbriae-mediated adhesion of Escherichia coli to human ileostomy glycoproteins was studied in vitro . Beta-lactoglobulin isolated from sterilized formulae was found to perform significantly less well than preparations from spray-dried formulae (p = 0.05) . Great heterogeneity was observed in the adhesion inhibitory capacity of beta-lactoglobulin isolated from UHT-treated formulae . Therefore, no significant difference was observed between UHT-treated and sterilized formulae or spray-dried formulae (p > 0.10) . It can be hypothesized that beta-lactoglobulin from spray-dried and some UHT-treated infant formulae may affect the colonization of mucous membranes by E . coli strains causing neonatal septicaemia and meningitis. Clin Exp Allergy, 1998 Apr, 28(4), 423 - 33 Preserved epitope-specific T cell activation by recombinant Bet v 1-MBP fusion proteins; van Neerven RJ et al.; BACKGROUND: Allergen-specific T cells play an important role in the allergic immune response, and are thought to be the principal target in specific immunotherapy . OBJECTIVE: The aim of the present study was to evaluate if fusion proteins of allergens with bacterial proteins can be used to activate and bias allergen-specific T cells, and to characterize T cell epitopes . METHODS: The complete gene of Bet v 1, the major birch pollen allergen, was amplified by PCR from birch pollen mRNA, and cloned in pKK223-3 . The complete gene or truncated sequences were transferred to pMAL-c and expressed in E . coli as fusion proteins with maltose binding protein (MBP) . The complete fusion protein, and the truncated fusion proteins were used for studies with Bet v 1-specific T cells . RESULTS: Bet v 1-specific T cells reacted similarly with purified and crude Bet v 1-MBP proteins . Therefore, crude preparations were used to study the epitope-specificity of 11 Bet v 1-specific T cell clones . Six distinct T cell epitopes were determined in this way . Interestingly, the T cell epitope of three T cell clones, that did not react with synthetic peptides in a previous study, was identified . In addition, the presence of MBP as a fusion partner to Bet v 1 was shown to influence TH2/TH1 cytokine production in T cell lines, but not in established T cell clones . CONCLUSION: Using crude preparations of recombinant fusion proteins of Bet v 1 with MBP, multiple T cell epitopes were identified in Bet v 1 . As T cell activation is preserved in this system, the generation of recombinant allergens with TH1-inducing proteins as fusion partners might be considered as a T-cell targeted approach for specific immunotherapy. Clin Rheumatol, 1998, 17(2), 144 - 7 Anti-transfer RNA antibodies in two patients with pulmonary fibrosis, Raynaud's phenomenon and polyarthritis; Ohosone Y et al.; Patient W.S . (a 61-year-old woman) and patient T.M . (a 41-year-old man) developed recurrent fevers, polyarthritis, Raynaud's phenomenon and interstitial pulmonary fibrosis without apparent polymyositis . From HeLa cell extracts, sera from both patients immunoprecipitated all species of intact and deproteinised tRNAs . To identify the antibody binding site more precisely, tRNAs transcribed in vitro from cloned Escherichia coli tRNA genes and various mutants were prepared and used as antigens for immunoprecipitation . When the TpsiC loop, or the D loop were deleted, such mutants were not bound by both sera, suggesting that the D and TpsiC loops were required for antibody binding . Abrogation of tRNA binding occurred when 18G of tRNATrp was replaced with 18A to break the tertiary L-shape structure of tRNA . These results strongly suggest that sera from W.S . and T.M . recognise the tertiary conformation of L-shaped tRNA which is constructed with both D and TpsiC loops . These autoantibodies may also serve as a marker for a new subset of patients with connective tissue diseases that is distinct from anti-aminoacyl-tRNA synthetase syndrome. Nephrol Dial Transplant, 1998 Jun, 13(6), 1458 - 64 Removal of cytokines and activated complement components in an experimental model of continuous plasma filtration coupled with sorbent adsorption; Tetta C et al.; BACKGROUND: Sepsis is associated with enhanced cytokine production . Here, we examined the in vitro removal of plasma cytokines during continuous plasmafiltration coupled with sorbent adsorption . METHODS: Proinflammatory (tumour necrosis factor-alpha, interleukins-1, -8) and anti-inflammatory (interleukin 1 receptor antagonist, soluble tumour necrosis factor receptor type I and II) cytokines in whole blood spiked with Escherichia coli endotoxin were determined during 2-h recirculation in the ultrafiltrate (condition A), plasma filtrate (condition B), before and after different sorbents (of the Amberlite-, Amberchrome- Ambersorb -type and charcoal) . We studied the maximal adsorbing capacity, the 1% leakage test for cytokines and C3a des Arg and the adsorption of complement-dependent leukocyte chemiluminescence . Plasma proteins eluted from the resins were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting with an anti-human alpha2-macroglobulin . RESULTS: In condition B, we observed a 40- and 121-fold % increase (vs condition A) in the removed mass and clearance of tumour necrosis factor-alpha . For all other cytokines, the removed mass and the clearance increased from 2.3- up to 6-fold . The Amberchrome but not the Amberlite or Ambersorb resins could remove the highest amount of cytokines and could reduce complement-dependent chemiluminescence . Two protein bands of approximately 400,000 D and 200,000 D were eluted only from Amberchrome resins and immunoprecipitated by anti-human alpha2-macroglobulin and anti-human C3c antibodies, respectively . CONCLUSIONS: These studies suggest an efficient removal of cytokines in continuous plasmafiltration with sorbent adsorption . The binding of alpha2-macroglobulin, a carrier of cytokines in plasma, might be a additional mechanism in the removal of cytokines from plasma. Planta, 1998 Jul, 205(3), 428 - 37 Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers; Geigenberger P et al.; Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa 1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U . Sonnewald, 1992, Plant J 2: 571-581) . The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L . cv . Desiree) tubers . Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa 1 transformants would restrict the mobilisation of sucrose and conversion to starch . Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa 1 tubers . Unexpectedly, the rate of degradation of {14C} sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20-30% in ppa 1 tubers compared to wild-type tubers . Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa 1 tubers were investigated . (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to three-fold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase . They also contained 30-100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase . (ii) The transformants contained higher pools of uridine nucleotides . As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP . (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold . In stored ppa 1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase . It is concluded that in growing tubers PPi has a wider-significance than just being an energy donor for specific reactions in the cytosol . Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein . Possible ways in which PPi hydrolysis could affect these processes are discussed. Southeast Asian J Trop Med Public Health, 1997, 28 Suppl 3, 164 - 6 Surface and total tissue factor activity of endothelial cells; Sangtawesin W et al.; With a technic that was developed by us, we found that normal human umbilical vein endothelial cells (HUVEC) in culture characteristically had very little tissue factor (TF) activity either on the surface or in the cells which had been disrupted . In the presence of endotoxin (E . coli O26:B6), a trigger for thrombosis in septicemic patients, we could not detect an increased TF activity of HUVEC on its surface . However, an increase in TF (total TF) was detected after disruption of the cells . The increase in total TF was dose-dependent . Endotoxin at the concentration of 10 micrograms/ml caused around 5 fold increase in total TF activity compared to that of HUVEC in the absence of endotoxin. J Chromatogr A, 1998 May 8, 806(1), 31 - 45 Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography; Prazeres DM et al.; Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy . Anion-exchange chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique . This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18 . NaCl gradient elution enabled the isolation of supercoiled plasmid from low-M(r) RNA, cDNA and plasmid variants . A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants . Anion-exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield . Supercoiled plasmid was recovered in a single fraction with a 62 +/- 8% yield . Loadings higher than 40 micrograms/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms . Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays. Wei Sheng Wu Xue Bao, 1996 Oct, 36(5), 344 - 50 {Studies on modulation and control sigma 38 and RMF for the expression of some genes}; Ding Q et al.; The E . coli mutants and wild type strains of rpoS and rmf were cultured in rich medium LB and limited component medium EP respectively . During the stationary phase, the viable cells of mutants were less than wild type strains's . The change of the product of serial proteins was quantified with Western blot . Sigma 38 has not effects on the product of rpoA, rpoB, rpoC, groE and tu gene, depress the transcription of crp and promote the expression of rmf . RMF can promote expression of rpoA, rpoD, groEl, rho, ompA and tufA gene in rich medium, but not in limited medium, and then depress and promote the expression of crp and rpoS respectively. Wei Sheng Wu Xue Bao, 1996 Dec, 36(6), 417 - 22 {Studies on midecamycin 4"-O-propionyltransferase gene structure}; Zhang X et al.; A BamHI-BamHI 8.0 kb DNA fragment which contains midecamycin propionyltransferase (mpt) gene was digested with different restriction enzymes and the restriction map was made . The mpt gene was localized in a EcoRI-EcoRI-PstI3.0 kb DNA fragment by Southern blot analysis using a 2.4 kb DNA fragment of the CarE gene as a probe . The 3.0 kb DNA fragment of mpt gene was cloned into E . coli/Streptomyces shuttle vector pWHM3 and a recombinant plasmid pWFPE was obtained . S . ambofaciens(pWFPE) and S . lividans(pWFPE) can convert endogenously synthesized or exogenously added spiramycin into 4"-O-propionylspiramycin, respectively . Sequence analysis of mpt gene demonstrated an open reading frame in the EcoRI-EcoRI-PstI3.0 kb DNA fragment, which starts with ATG and ends with TGA . Mpt gene encodes a product of 388 aa . G+C mol% of mpt is 68.0 and G+C mol% of 3rd codon position is 91.5 . The putative product of mpt has a identity of 67.6% and a similarity of 86.4% with CarE product . A consensus RBS GAGGT in the 6bp upstream from ATG and a promoter region were found . An inverted repeat sequence in the downstream from TGA acts as transcriptional terminator. Mutat Res, 1998 Jun 5, 401(1-2), 179 - 91 High and low UV-dose responses in SOS-induction of the precise excision of transposons tn1, Tn5 and Tn10 in Escherichia coli; Aleshkin GI et al.; UV-inducible precise excision of transposons is a specific SOS-mutagenesis process . It deals with the deletion formation which has previously been demonstrated to involve direct or inverted IS-sequences of transposons . The process was used for revisiting the targeted and untargeted SOS-mutability and its relationship to the key genes for SOS-mutagenesis: the recA, lexA and umuDC . The precise excision of transposons Tn5 and Tn10 from the chromosomal insertion sites ade128 and cyc750 is induced in Escherichia coli K-12 and B cells, wild-type for DNA-repair, both by the low doses of UV-light ranging from 0.25 J m-2 to 2.5 J m-2 and the high doses within the range 5.0-40.0 J m-2 . Precise excision of these transposons induced by the range of low doses incapable to induce targeted point mutations reveals its mostly untargeted nature . This process for the transposon Tn1 is not induced by UV-light within the range of doses 0.25-2.5 J m-2 while its induction is possible by UV-fluences ranging from 5.0 to 40.0 J m-2 . A dose-response of the precise excision of Tn1 is similar to that of the UV-induced reversion of trpUAA point mutation that is targeted by nature and contrasts to the UV-inducible precise excision of Tn5 and Tn10 . Both types of UV-inducible precise excision, demonstrated either by Tn1 or Tn5 and Tn10, are eliminated by mutations in the lexA, recA and umuDC genes indispensable for UV-induced SOS-mutability . The palindromic structures different for the transposons Tn1, Tn5 and Tn10 are discussed to be involved and affect the targeted and untargeted precise excision of transposons induced by UV-light . Mutat Res, 1998 Jun 5, 401(1-2), 165 - 78 Comparison of in vivo mutagenesis in the endogenous Hprt gene and the lacI transgene of Big Blue(R) rats treated with 7, 12-dimethylbenz{a}anthracene; Manjanatha MG et al.; The lacI transgene of Big Blue(R) (BB) rats was evaluated as a reporter of in vivo mutation by comparing mutant frequencies (MFs) in it and in the endogenous Hprt gene . Seven-week old female BB rats were given single doses of 0, 20, 75 and 130 mg/kg of 7, 12-dimethylbenz(a)anthracene (DMBA) by gavage, and Hprt and lacI MFs in splenic lymphocytes were measured over a period of 18 weeks . The Hprt MFs in treated rats increased for 10 weeks and then declined; 130 mg/kg of DMBA produced a maximum Hprt MF of 168+/-11.4x10-6 clonable lymphocytes, while the MF in control rats was 7.4+/-1 . 5x10-6 . DMBA exposure of generic F344 rats resulted in a similar time-course of mutant induction but produced about 50% higher Hprt MFs with the 75 and 130 mg/kg doses . In contrast, the lacI MFs increased for 6 weeks and then remained relatively constant; 130 mg/kg of DMBA produced a maximum increase in lacI MF of 341+/-83x10-6 PFU compared with 25+/-5x10-6 PFU in control rats . The Hprt mutant frequencies in DMBA-treated BB and F344 rats were significantly increased over control values for every dose-time combination examined, while only the 130 mg/kg dose consistently produced lacI MFs that were significantly above the controls . In addition, the fold-increase in MF for treated vs . control rats was two times higher for the Hprt gene than the lacI gene due to the higher MFs in the lacI gene of control rats . Differences between the lacI and Hprt genes in the kinetics of mutant induction, in the frequency of induced mutants, and in the sensitivity of mutant detection could be explained at least partially by the properties of these two genes . Mutat Res, 1998 Jun 5, 401(1-2), 153 - 64 Comparative mutational spectra of the nitrogen mustard chlorambucil and its half-mustard analogue in Chinese hamster AS52 cells; Yaghi BM et al.; Nitrogen mustards play an important role in current cancer chemotherapy . The most effective antitumour agents are those carrying two alkylating functions, probably through their ability to form interstrand cross-links in DNA . Such lesions appear to create more of a block in DNA replication and are more difficult to repair than are most monoadducts . Although there were early reports that monofunctional drugs were more mutagenic than the bifunctional drugs, this has not been formally proved using structurally related drugs in a mutagenicity assay capable of detecting a range of different events . We have studied both the mutagenic potency and spectrum of events caused by treatment with the clinical agent, chlorambucil, compared with its half-mustard analogue, in Chinese hamster ovary (CHO)-AS52 cells . Although both drugs caused comparable increases in mutation frequency at doses killing 90% of cells (from around 9x10-6 to around 9x10-5 mutant cells), the nature of events differed significantly between the drugs . By far the majority of mutations caused by the half-mustard were transversion mutations, and almost all of these could be interpreted in relation to the DNA adducts that are known to be formed . In contrast, the majority of chlorambucil-induced mutations were major deletions, and point mutations were only identified from a few clones . Parallel micronucleus assays verified that chlorambucil has a stronger ability to break chromosomes than the half-mustard . These two drugs are thought to form similar monoadducts, but only the full mustard can form interstrand cross-links . The data suggest that DNA cross-links, although only a minor fraction of the total lesions, dominate the mutagenic spectrum and lead to gross changes at the chromosome level that can not be readily associated with individual lesions produced by the drug . Mutat Res, 1998 Jun 5, 401(1-2), 99 - 110 The in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene; Recio L et al.; 1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice . We determined the lacI mutant frequency and mutational spectrum in spleen following inhalation exposure to BD at levels that are known to induce tumors . B6C3F1 lacI transgenic mice were exposed to air or to 62.5, 625, or 1250 ppm BD for 4 weeks (6 h/day, 5 days/week) and euthanized 14 days after the last exposure . BD increased the lacI mutant frequency in spleen at all levels of BD examined . In BD-exposed mice, an increased frequency of G:C-->A:T transitions occurred at non-5'-CpG-3' sites . Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs when compared to air controls . The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum . These data demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissue-specific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors . Mutat Res, 1998 Mar 16, 413(2), 95 - 101 New Escherichia coli WP2 tester strains highly sensitive to reversion by oxidative mutagens; Blanco M et al.; New Escherichia coli strains have been added to the WP2 mutagenicity test for the specific detection of oxidative mutagens . Strain IC203 derives from WP2 uvrA/pKM101 and is highly sensitive to oxidative stress due to a deficiency in the OxyR function . Following exposure to t-butyl hydroperoxide (BuOOH) or menadione (MD), but not to 4-nitroquinoline 1-oxide (4NQO), strain IC203 (oxyR) shows increased mutability with respect to the oxyR+ parent . The advantage that the OxyR deficiency confers on IC203 strain in detecting oxidative mutagens is not obtained with strains deficient in either katG or ahpCF, two OxyR-regulated genes . Strain IC206, a derivative of WP2 uvrA carrying a deletion of the umuDC genes and deficient in the MutY glycosylase, has also been added to the WP2 test for the detection of SOS-independent mutations promoted by 8-oxoguanine lesions . Induction of these mutations was observed after treatment with BuOOH, but not after MD or 4NQO exposure . The two new strains, IC203 and IC206, can be useful for the screening of mutations resulting from oxidative stress as well as in studies on antioxidants preventing mutagenesis . Arch Microbiol, 1998 Jul, 170(1), 50 - 8 Two membrane anchors of Wolinella succinogenes hydrogenase and their function in fumarate and polysulfide respiration; Gross R et al.; Wolinella succinogenes can grow by anaerobic respiration with fumarate or polysulfide as the terminal electron acceptor, and H2 or formate as the electron donor . A DeltahydABC mutant lacking the hydrogenase structural genes did not grow with H2 and either fumarate or polysulfide . In contrast to the wild-type strain, the mutant grown with fumarate and with formate instead of H2 did not catalyze the reduction of fumarate, polysulfide, dimethylnaphthoquinone, or benzyl viologen by H2 . Growth and enzymic activities were restored upon integration of a plasmid carrying hydABC into the genome of the DeltahydABC mutant . The DeltahydABC mutant was complemented with hydABC operons modified by artificial stop codons in hydA (StopA) or at the 5'-end of hydC (StopC) . The StopC mutant lacked HydC, and the hydrophobic C-terminus of HydA was missing in the hydrogenase of the StopA mutant . The two mutants catalyzed benzyl viologen reduction by H2 . The enzyme activity was located in the membrane of the mutants . A mutant with both modifications (StopAC) contained the activity in the periplasm . The three mutants did not grow with H2 and either fumarate or polysulfide, and did not catalyze dimethylnaphthoquinone reduction by H2 . We conclude that the same hydrogenase serves in the anaerobic respiration with fumarate and with polysulfide . HydC and the C-terminus of HydA appear to be required for both routes of electron transport and for dimethylnaphthoquinone reduction by H2 . The hydrogenase is anchored in the membrane by HydC and by the C-terminus of HydA . The catalytic subunit HydB is oriented towards the periplasmic side of the membrane. Biochem J, 1998 Jul 1, 333 ( Pt 1), 183 - 91 A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity; Dennis CA et al.; We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude . In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9 . Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase . Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned . Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further . Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer . This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity. Biochem J, 1998 Jul 1, 333 ( Pt 1), 85 - 90 Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition; Green A et al.; Zinc fingers are among the major structural motifs found in proteins that are involved in eukaryotic gene regulation . Many of these zinc-finger domains are involved in DNA binding . This study investigated whether the zinc-co-ordinating (Cys)2(His)2 motif found in the three zinc fingers of zif268 could be replaced by a (Cys)4 motif while still preserving DNA recognition . (Cys)2(His)2-to-(Cys)4 mutations were generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together . Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finger 2 renders the polypeptide incapable of DNA recognition . The protein-DNA interaction was examined in greater detail by using a methylation-interference assay . The mutant polypeptides containing the (Cys)4 motif in fingers 1 or 3 recognize DNA in a manner identical to the wild-type protein, suggesting that the (Cys)4 motif appears to give rise to a properly folded finger . Additional results indicate that a zif268 variant containing a (Cys)2(His)(Ala) arrangement in finger 1 is also capable of DNA recognition in a manner identical to the wild-type polypeptide . This appears to be the first time that such alterations, in the context of an intact DNA-binding domain, have still allowed for specific DNA recognition . Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA-binding by zinc fingers. Biochem J, 1998 Jul 1, 333 ( Pt 1), 33 - 43 Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms; Hughes RK et al.; The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L . cv . Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli . A comparison of both isoforms purified to apparent homogeneity from E . coli and pea seeds has confirmed the authenticity of the recombinant products and established the properties of the native enzymes . Despite 86% similarity at the amino acid sequence level, the enzymes have distinct properties . They have been characterized in terms of specific activity, Fe content, optimum pH, substrate and product specificity, apparent Km and Vmax for the preferred substrate, linoleic acid, and interfacial behaviour with linoleic acid . We have used this evidence, in addition to EPR spectroscopy of the hydroperoxide-activated enzymes and estimates of kcat/Km, to propose different reaction mechanisms for linoleic acid oxidation for the two isoforms . The differences relate primarily to carbonyl production from linoleic acid for which we propose a mechanism . This implicates the release of a peroxyl radical in an aerobic hydroperoxidase reaction, as the source of the carbonyl compounds formed by dismutation of the liberated peroxyl radical. Biochem J, 1998 Jul 1, 333 ( Pt 1), 27 - 32 Functional characterization of a recombinant form of the C-terminal, globular head region of the B-chain of human serum complement protein, C1q; Kishore U et al.; The first step in the activation of the classical pathway of the complement system by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM . The globular heads of C1q are located C-terminal to the six triple-helical stalks present in the molecule; each head is considered to be composed of the C-terminal halves (3x136 residues) of one A-, one B- and one C-chain . It is not known if the C-terminal globular regions, present in each of the three types of chain, are independently folded modules (with each chain having distinct binding properties towards immunoglobulins) or whether the different binding functions of C1q are dependent upon a globular structure which relies on contributions from all three chains . As a first step towards addressing this question, we have expressed the globular head region (residues 87-226) of the C1q B-chain (ghB) as a soluble fusion protein with maltose-binding protein (MBP) in Escherichia coli . The affinity purified fusion protein, designated MBP-ghB, behaved as a dimer on gel filtration and bound preferentially to aggregated IgG rather than to IgM . It could also inhibit C1q-dependent haemolysis of both IgG- and IgM-sensitized erythrocytes . After its release from MBP, by use of Factor Xa, the free ghB exhibited a tendency to aggregate and come out of solution . Since MBP is known to be a monomeric molecule, the dimerization of the MBP-ghB fusion polypeptide is probably brought about by the ghB region, perhaps through hydrophobic interactions within the ghB region . The functional behaviour of MBP-ghB indicates that the globular regions of C1q may adopt a modular organization, i.e . each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. Biochem J, 1998 Jul 1, 333 ( Pt 1), 5 - 9 The majority of human glutathione peroxidase type 5 (GPX5) transcripts are incorrectly spliced: implications for the role of GPX5 in the male reproductive tract; Hall L et al.; An epididymis-specific, secretory glutathione peroxidase (GPX5) has been proposed previously to play a role in protecting mammalian sperm membranes from the deleterious effects of lipid peroxidation, which, if not contained, can lead to reduced fertilizing capacity . Here we report the cDNA cloning of human GPX5 and show that the majority of transcripts contain a 118 nt frame-shifting deletion, arising, most likely, from inappropriate excision of exon 3 during processing . Antisera raised against recombinant human GPX5 cross-reacted with rat and macaque (Macaca fascicularis) epididymal proteins of the size expected for full-length, active GPX5 . However, no similar reactivity could be demonstrated in any of the human samples tested. Calcif Tissue Int, 1998 Jul, 63(1), 63 - 6 Histopathological study of the role of CD4- and CD8-positive T cells on bone resorption induced by Escherichia coli endotoxin; Hara Y et al.; The purpose of this study was to clarify the involvement of CD4+ and CD8+ T cells on bone resorption induced by Escherichia coli endotoxin . Two kinds of monoclonal antibodies, anti-CD4 and/or anti-CD8, were employed for the depletion of each or both T cell subsets . E . coli endotoxin was injected into mouse mesial gingiva of the first molar of the left mandible every 48 hours for up to 14 days (7 injections) . The mice were divided into four groups: CD4-depleted, CD8-depleted, T cell-depleted, and normal . The mice were sacrificed on the day after the 1st, 3rd, 4th, 5th, and 7th injection and alveolar bone was examined histopathologically and histomorphometrically . Bone surface in contact with osteoclasts was defined as the site of active resorption and the ratios of active resorption were compared among the four groups . In addition, sections obtained after the 1st, 4th, and 7th injection were immunohistologically stained in order to confirm the presence or absence of CD4+ or CD8+ T cells . Alveolar bone resorption gradually increased in normal mice as the number of injections increased . In contrast, alveolar bone resorption was significantly weaker in each or both subset-depleted mice . For the duration of the experimental period, the number of CD4+ T cells in CD8-depleted and normal mice significantly increased with increasing bone resorption . Considering the function of CD4+ and CD8+ T cells, these results suggest that each subset preferentially acts as a macrophage activator in the early period of bone resorption induced by E . coli endotoxin. EMBO J, 1998 Jun 1, 17(11), 3207 - 16 Inhibition of Escherichia coli RecA coprotease activities by DinI; Yasuda T et al.; In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions . The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA . In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene . dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids . Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation . In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells . Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type . Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD. EMBO J, 1998 Jun 1, 17(11), 3188 - 96 Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches; Felden B et al.; Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs . To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da . Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass . Also, chemical modifications were used to locate mass-silent modifications . The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine . Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs . Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar . A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation. Biochem J, 1998 Jun 1, 332 ( Pt 2), 413 - 9 Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator; Macheroux P et al.; Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA) . CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly alpha-helical . The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators . The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA . In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant . Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module . The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem . Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant . Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen . We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly. Zentralbl Bakteriol, 1998 May, 287(4), 461 - 73 Binding of immobilized fibronectin by biliary drain isolates; Yu JL et al.; Occlusion of biliary stents, as the result of bacterial adhesion and colonization onto biliary stents, still remains a major problem . Biliary proteins, such as fibronectin (Fn) and vitronectin (Vn), have been presumed to be involved in the process of bacterial adhesion to biliary biomaterial . In the present study, Fn binding by 5 strains of E . coli isolated from biliary drains or from bile was studied . All strains did not bind detectable amounts of soluble Fn but bound to immobilized plasma Fn . Adhesion of four strains of E . coli to ovalbumin was reduced by periodate treatment of ovalbumin, but adhesion to Fn was unaffected . Adhesion was inhibited by mannose-containing saccharides, trypsin treatment of the protein, and protease treatment of the bacterial cells . Autoradiography showed that components of cell extracts from three E . coli strains bind 125I-Fn but not a 150 kD Fn fragment . The findings indicate that the adhesion of these bacteria to Fn is a protein-protein interaction, inhibited by D-mannose, and possibly mediated by fimbrial components. Infect Immun, 1998 Jul, 66(7), 3480 - 4 Inhibition of class II major histocompatibility complex antigen processing by Escherichia coli heat-labile enterotoxin requires an enzymatically active A subunit; Matousek MP et al.; Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing . Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes . Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity. Placenta, 1998 May, 19(4), 307 - 14 Culture of human amniotic cells: a system to study interferon production; Carvalho AF et al.; This study investigated whether primary culture of human amniotic membrane cells (PCHAM) could be used as an in vitro model system for the study of interferon (IFN) production . PCHAM cells infected with Newcastle disease virus (NDV) produced the two antigenic types of IFN, previously shown in a amniotic membrane cells (HAM) system . PCHAM IFN was detected as early as 2 h after NDV infection and was composed by two antigenically distinct fractions, one neutralized with anti-HuIFN beta antibody and another that is not related to IFN beta, -alpha and -gamma . These fractions correspond respectively to 80 and 20 per cent of the IFN produced 4 h after virus induction, 55 and 45 per cent of the IFN produced from 4 to 12 h and 67 and 33 per cent of the IFN produced 12 h after virus induction . A cDNA library, established from PCHAM with or without NDV infection, was screened for IFN alpha and -beta using specific primers . The PCR product, amplified by IFN beta primers, was cloned, sequenced and expressed in Escherichia coli M15 . The sequences of several cloned cDNAs were identical to HuIFN beta gene and the antiviral activity of the expressed protein was neutralized only by antiHuIFN-beta antibody . The other IFN fraction not neutralized by polyclonal antibodies anti-IFN beta, -alpha and -gamma is now being studied. Neuropeptides, 1998 Apr, 32(2), 191 - 6 Identification of the spinal degradation products and inhibition of adenylate cyclase by recombinant rat galanin message-associated peptide; Andell-Jonsson S et al.; In rat preprogalanin, galanin is C-terminally flanked by a 60 amino acid long peptide: galanin message-associated peptide (GMAP) . GMAP sequences in different species show high degree of homology, suggesting a biological role . However, the study of the physiological and pharmacological actions of this peptide have been hampered by lack of availability of this large peptide, its fragments and well-characterized antibodies to GMAP . This study report the production of GMAP in Escherichia coli and the use of the recombinant peptide to define its degradation products in the spinal cord . The GMAP fragments formed upon incubation of GMAP with membranes of lumbar spinal cord were identified by sequencing and were also produced by solid phase synthesis for studies on second messenger systems . Furthermore, the study demonstrates that GMAP inhibits forskolin stimulated adenylate cyclase activity in a concentration dependent manner, while GMAP and its synthetic fragments did not affect cGMP level. Electrophoresis, 1998 May, 19(6), 901 - 8 Sample handling for proteome analysis; Staudenmann W et al.; The main factor limiting the sensitivity range for the identification of proteins isolated by two-dimensional (2-D) electrophoresis is sample handling: protein detection limits on the gel, losses during extraction and digestion, as well as interference of gel contaminants and detergents with the mass spectrometry (MS) detection increasing background noise . At the one hundred picomole level, losses are fairly negligible but when the amounts drop below 1 picomole (and subfemtomole peptide detection limits have been reported recently by MS), the losses become a critical point . In order to extend proteome analysis to include very low copy number proteins, methods must be developed to minimize losses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise . We present several methods that we have developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)-free form which does not require staining, (ii) increase protein extraction and digestion yields and lower the contamination by autoproteolytic products, and (iii) allow direct modification of the peptide mixture to generate sequence tags. Arch Virol, 1998, 143(4), 769 - 80 Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus; Naidu RA et al.; Two strains of whitefly-transmitted cowpea mild mottle virus (CPMMV) causing severe (CPMMV-S) and mild (CPMMV-M) disease symptoms in peanuts were collected from two distinct agro-ecological zones in India . The host-range of these strains was restricted to Leguminosae and Chenopodiaceae, and each could be distinguished on the basis of symptoms incited in different hosts . The 3'-terminal 2500 nucleotide sequence of the genomic RNA of both the strains was 70% identical and contains five open reading frames (ORFs) . The first three (P25, P12 and P7) overlap to form a triple gene block of proteins, P32 encodes the coat protein, followed by P12 protein located at the 3' end of the genome . Genome organization and pair-wise comparisons of amino acid sequences of proteins encoded by these ORFs with corresponding proteins of known carlaviruses and potexviruses suggest that CPMMV-S and CPMMV-M are closely related to viruses in the genus Carlavirus . Based on the data, it is concluded that CPMMV is a distinct species in the genus Carlavirus. Vet J, 1998 May, 155(3), 263 - 74 Detrimental effects on villus form during conventional oral rehydration therapy for diarrhoea in calves; alleviation by a nutrient oral rehydration solution containing glutamine; Brooks HW et al.; This paper examines the possibility that treatment of diarrhoea with conventional oral rehydration solutions (ORSs) may be detrimental to villus structure by imposing nutrient deprivation and that such detrimental effects may be reduced or avoided by using a nutrient ORS . A conventional WHO-type ORS (W) was compared with two nutrient solutions (N and G) both containing high glucose concentrations and the latter containing glutamine; their effects on enteric structure were assessed by morphometric analysis of samples obtained from diarrhoeic calves after 96 h treatment . Comparisons were also made with samples from controls and diarrhoeic calves at the stage where oral rehydration would have begun in the treated groups . As in our previous ORS studies, diarrhoea was induced with enterotoxigenic Escherichia coli (09:K30:K99) . We measured villus length and width, crypt depth and width and calculated villus surface area in proximal, mid and distal small intestine (PSI, MSI, DSI), using standard morphometric techniques . Proximal and distal spiral colon samples (PC, DC) were examined for crypt depth and width; mitoses per crypt were counted in samples from all regions . Non-diarrhoeic calves showed the expected gradient of villus length through PSI, MSI and DSI, hence data for each region are normalized as a percentage of the control value for that region . PSI showed the greatest loss of villus length and surface area (50%) with diarrhoea . In MSI and DSI the villus loss was greater with solution W and N or G, as were increased mitoses and crypt depth . Crypt depth and mitoses also increased in the colon with solution W . Colonic crypt width increased with diarrhoea and conventional oral rehydration but less so with G; there is reason to believe that such changes have functional significance . Crypt changes in colon, MSI and DSI were least with solution G . The changes developing in diarrhoeic calves prior to treatment were thus less apparent in those treated with a nutritional ORS, particularly if it contained glutamine. Curr Biol, 1998 Jun 4, 8(12), 725 - 7 Formation of RuvABC-Holliday junction complexes in vitro; Davies AA et al.; In Escherichia coli, the RuvA, RuvB and RuvC proteins are required for the late stages of homologous recombination and DNA repair . RuvA and RuvB form a complex that interacts with Holliday junctions--crossed DNA structures that are recombination intermediates--and promotes branch migration; RuvC is a junction-specific endonuclease that resolves Holliday junctions and completes the recombination process . Because genetic and biochemical experiments suggest that the processes of branch migration and resolution are linked, coimmunoprecipitation experiments were carried out to determine whether the three Ruv proteins interact to form a functional complex (RuvABC) . Using a synthetic Holliday junction, a multisubunit complex containing the junction and RuvA, RuvB and RuvC was detected . In the absence of RuvB, RuvAC-junction complexes were observed . Complex formation was not facilitated by duplex DNA . The identification of a RuvABC-junction complex provides direct evidence that the RuvABC proteins interact at the Holliday junction. Arch Biochem Biophys, 1998 Jun 15, 354(2), 215 - 24 Role of allosteric: zinc interdomain region of the regulatory subunit in the allosteric regulation of aspartate transcarbamoylase from Escherichia coli; Rastogi VK et al.; The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme . The present work shows that this interface also affects CTP and CTP-UTP inhibition and proposes a structural explanation for the effects . Mutant enzymes derived from nonselective mutagenesis of residues r101-r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects . The cooperative behavior of the enzymes was affected, as indicated by reduced aspartate S0.5 values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C . In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants . The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP . Finally, the I103T mutant enzyme had an increased S0.5 value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced . Most of these allosteric variations can be explained in terms of perturbations to the "tongue and groove" hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface ("reg1:cat4") between regulatory and catalytic subunits . Mol Cell Biol, 1998 Jul, 18(7), 4079 - 88 Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor; Pongratz I et al.; The dioxin receptor is a ligand-regulated transcription factor that mediates signal transduction by dioxin and related environmental pollutants . The receptor belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of factors, which, in addition to the bHLH motif, contain a PAS region of homology . Upon activation, the dioxin receptor dimerizes with the bHLH-PAS factor Arnt, enabling the receptor to recognize xenobiotic response elements in the vicinity of target genes . We have studied the role of the PAS domain in dimerization and DNA binding specificity of the dioxin receptor and Arnt by monitoring the abilities of the individual bHLH domains and different bHLH-PAS fragments to dimerize and bind DNA in vitro and recognize target genes in vivo . The minimal bHLH domain of the dioxin receptor formed homodimeric complexes, heterodimerized with full-length Arnt, and together with Arnt was sufficient for recognition of target DNA in vitro and in vivo . In a similar fashion, only the bHLH domain of Arnt was necessary for DNA binding specificity in the presence of the dioxin receptor bHLH domain . Moreover, the bHLH domain of the dioxin receptor displayed a broad dimerization potential, as manifested by complex formation with, e.g . , the unrelated bHLH-Zip transcription factor USF . In contrast, a construct spanning the dioxin receptor bHLH domain and an N-terminal portion of the PAS domain failed to form homodimers and was capable of dimerizing only with Arnt . Thus, the PAS domain is essential to confer dimerization specificity of the dioxin receptor. Mol Cell Biol, 1998 Jul, 18(7), 3771 - 81 Even-skipped represses transcription by binding TATA binding protein and blocking the TFIID-TATA box interaction; Li C et al.; The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery . Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter . We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II . In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression . We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression . This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain . Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP . Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding . Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction . Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide . We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter. Infect Immun, 1998 Jul, 66(7), 3470 - 5 Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas; Noormohammadi AH et al.; Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes . A single gene (vlhA) from M . synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli . The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA . Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in {3H}palmitate-labelled membrane proteins . Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family . The vlhA gene was shown to hybridize to multiple restriction fragments of the M . synoviae genome, suggesting that it was also a member of a multigene family . These findings indicate that coordinate phase variation of the two major surface antigens of M . synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas . The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally. Infect Immun, 1998 Jul, 66(7), 3384 - 9 Cytotoxicity of hemolytic, cytotoxic necrotizing factor 1-positive and -negative Escherichia coli to human T24 bladder cells; Island MD et al.; Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types . A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates . To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1(+), and CNF1(-) cystitis strains toward human T24 bladder epithelial cells in vitro . A total of 29 isolates from two collections of cystitis-associated E . coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain . Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation . As a group, CNF1(+) isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1(-) strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains) . To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E . coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1 . Compared to the CNF1(+) parental isolates, no change in cytotoxicity was detected in these cnf1 mutants . Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E . coli to damage human bladder cell monolayers in vitro. Infect Immun, 1998 Jul, 66(7), 3311 - 6 Intestinal immune responses to an inactivated oral enterotoxigenic Escherichia coli vaccine and associated immunoglobulin A responses in blood; Ahren C et al.; An inactivated oral enterotoxigenic Escherichia coli (ETEC) vaccine against ETEC diarrhea was given to 25 adult Swedish volunteers . The vaccine consisted of formalin-killed E . coli bacteria expressing the most common colonization factor antigens (CFAs), i.e., CFA/I, -II, and -IV, and recombinantly produced cholera B subunit (CTB) . Immunoglobulin A (IgA) antibody responses in intestinal lavage fluid to CTB and CFAs were determined and compared with corresponding responses in stool extracts and serum as well as with IgA antibody-secreting cell (ASC) responses in peripheral blood . Two doses of vaccine induced significant IgA responses to the different CFAs in lavage fluid in 61 to 87% of the vaccinees and in stool in 38 to 81% of them . The most frequent responses were seen against CFA/I . The magnitudes of the antibody responses against CTB and CFA/I in stool correlated significantly (CTB, P < 0.01; CFA/I, P < 0 . 05) with those in intestinal lavage . Intestinal lavage responses against CFAs were best reflected by the ASC responses, with the sensitivity of the ASC assay being 80 to 85%, followed by stool (sensitivity of 50 to 88%) and serum antibody (sensitivity of 7 to 65%) analyses . CTB-specific immune responses were seen in >90% of the vaccinees in all assays. Infect Immun, 1998 Jul, 66(7), 3270 - 8 Protective roles of gamma delta T cells and interleukin-15 in Escherichia coli infection in mice; Takano M et al.; The number of gamma delta T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice . The gamma delta T cells preferentially expressed invariant Vgamma6 and Vdelta1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS . Mice depleted of gamma delta T cells by T-cell receptor delta gene mutation showed impaired resistance against E . coli as assessed by bacterial growth . Macrophages from C3H/HeN mice infected with E . coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice . Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of gamma delta T cells in C3H/HeN mice after E . coli infection and diminished the host defense against the infection . These results suggest that LPS-stimulated gamma delta T cells play an important role in the host defense against E . coli infection and that IL-15 may be partly involved in the protection via an increase in the gamma delta T cells. Infect Immun, 1998 Jul, 66(7), 3155 - 63 Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli; Eslava C et al.; Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness . Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described . Work from our laboratories has implicated a ca . 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber . Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin . The toxin (designated the plasmid-encoded toxin {Pet}) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042 . Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a beta-barrel pore in the bacterial outer membrane and through which the mature protein is transported . The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E . coli and to EspC of enteropathogenic E . coli, an as yet cryptic protein . In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins . We hypothesize that other closely related members of this class may also produce enterotoxic effects. Infect Immun, 1998 Jul, 66(7), 3149 - 54 In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli; Navarro-Garcia F et al.; The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated . In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers . The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose . This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance . These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC . Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect . Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species . Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber . The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not . A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc . Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses . Our data suggest that some EAggEC strains produce a ca . 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid. Infect Immun, 1998 Jul, 66(7), 3043 - 9 Heterogeneous expression and release of CD14 by human gingival fibroblasts: characterization and CD14-mediated interleukin-8 secretion in response to lipopolysaccharide; Sugawara S et al.; To identify the role in periodontal inflammatory diseases of human gingival fibroblasts (HGF), the major constituents of gingival tissue, the expression of CD14, a possible lipopolysaccharide (LPS) receptor, and the release of soluble CD14 (sCD14) by HGF were examined . Among the HGF samples from the nine donors tested, more than 50% of the HGF from five donors expressed CD14 but less than 20% of HGF from the other four donors did so, as determined by flow cytometric analysis . The CD14 expression on the cell surface was correlated with the expression of CD14 mRNA . The HGF and skin and lung fibroblasts tested expressed no CD18, which indicates that fibroblasts do not possess other LPS receptors, such as CD11b/CD18 and CD11c/CD18 . The CD14 expression by the HGF was decreased after subculturing and was highest at the confluent stage of culture . The treatment of high-CD14-expressing (CD14(high)) HGF with phosphatidylinositol-phospholipase C reduced CD14 expression; this result and the increase in a 55-kDa CD14 indicate that the membrane CD14 (mCD14) on the HGF may be a 55-kDa glycosylphosphatidylinositol-anchored protein . CD14(high) HGF spontaneously released 48- and 57-kDa sCD14 . The total release of sCD14 by the HGF was augmented by gamma interferon and Escherichia coli LPS in accordance with the increased expression of mCD14 . The CD14(high) HGF secreted interleukin-8 in response to LPS, and the secretion was completely inhibited by anti-CD14 antibody . These results suggest that (i) HGF consist of populations that are heterogeneous on the basis of different levels of expression of CD14 and (ii) CD14(high) HGF secrete inflammatory cytokines in response to LPS via CD14. Plant Physiol, 1998 Jun, 117(2), 593 - 8 A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed; Cahoon EB et al.; Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Delta9) plus cis-vaccenic acid (18:1Delta11) in its seed oil . To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds . The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Delta9-18:0)-ACP desaturase from castor (Ricinis communis L.) and other species . Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Delta9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP . Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Delta9-18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases . Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Delta9-18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP . The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Delta9-18:0-ACP desaturase. Plant Physiol, 1998 Jun, 117(2), 559 - 63 Function and substrate specificity of the gibberellin 3beta-hydroxylase encoded by the Arabidopsis GA4 gene; Williams J et al.; cDNA corresponding to the GA4 gene of Arabidopsis thaliana L . (Heynh . ) was expressed in Escherichia coli, from which cell lysates converted {14C}gibberellin (GA)9 and {14C}GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3beta-hydroxylase . GA9 was the preferred substrate, with a Michaelis value of 1 microm compared with 15 microm for GA20 . Hydroxylation of these GAs was regiospecific, with no indication of 2beta-hydroxylation or 2,3-desaturation . The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated . In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs . Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively . The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15 . In contrast to the free acid, GA25 19,20-anhydride was 3beta-hydroxylated to give GA13 . 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2, 3-oxido-GA9 and GA6. Plant Physiol, 1998 Jun, 117(2), 533 - 43 Tissue culture-specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase; Song HS et al.; A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) alpha-subunit gene, ASA2, has been isolated from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell line (AB15-12-1) . The ASA2 cDNA contains a putative transit peptide sequence, and Southern hybridization shows that more than one closely related sequence is present in the tobacco genome . The ASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 microm 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity . The 5MTr was lost when the E . coli strain was transformed with an ASA2 site-directed mutant (phenylalanine-107-arginine-108 --> serine-107-glutamine-108) . Identical nucleotide sequences encoding the phenylalanine-107-arginine-108 region have been found in polymerase chain reaction-amplified 326-bp ASA2 genomic fragments of wild-type (5-methyl-tryptophan-sensitive {5MTs}) tobacco and a progenitor species . High-level ASA2 transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants . Promoter studies indicate that tissue specificity of ASA2 is controlled by the promoter region between -2252 and -607 . Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTs lines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression. Plant Physiol, 1998 Jun, 117(2), 455 - 64 Identification of the gene encoding the tryptophan synthase beta-subunit from Chlamydomonas reinhardtii; Palombella AL et al.; We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the beta-subunit of tryptophan synthase (TSB) . This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli . Hybridization analysis indicated that the gene exists in a single copy . The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms . With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole . These mutations fell into three complementation groups, MAA2, MAA7, and TAR1 . In vitro assays showed that mutations at each of these loci affected TSB activity . Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB . MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product. Plant Physiol, 1998 Jun, 117(2), 425 - 35 Characterization of SU1 isoamylase, a determinant of storage starch structure in maize; Rahman A et al.; Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm . Homozygous su1- mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin . These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis . SU1 is similar in sequence to alpha-(1-->6) glucan hydrolases (starch-debranching enzymes {DBEs}) . Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis . Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity . Two biochemical assays confirmed that SU1 hydrolyzes alpha-(1-->6) linkages in branched polysaccharides . Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase . Previous studies had shown, however, that su1- mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme) . Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1- mutant kernels . These data support the hypothesis that DBEs are directly involved in starch biosynthesis. Plant Physiol, 1998 Jun, 117(2), 417 - 23 A gene coding for tomato fruit beta-galactosidase II is expressed during fruit ripening . Cloning, characterization, and expression pattern; Smith DL et al.; beta-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, nonreducing beta-D-galactosyl residues from beta-D-galactosides . Several beta-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing beta(1-->4)-D-galactan . Although beta-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan, has been purified, cloning of the corresponding gene has been elusive . We report here the cloning of a cDNA, pTombetagal 4 (accession no . AF020390), corresponding to beta-galactosidase II, and show that its corresponding gene is expressed during fruit ripening . Northern-blot analysis revealed that the beta-galactosidase II gene transcript was detectable at the breaker stage of ripeness, maximum at the turning stage, and present at decreasing levels during the later stages of normal tomato fruit ripening . At the turning stage of ripeness, the transcript was present in all fruit tissues and was highest in the outermost tissues (including the peel) . Confirmation that pTombetagal 4 codes for beta-galactosidase II was derived from matching protein and deduced amino acid sequences . Furthermore, analysis of the deduced amino acid sequence of pTombetagal 4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader-sequence cleavage site, and three possible N-glycosylation sites . The predicted molecular mass and isoelectric point of the pTombetagal 4-encoded mature protein were similar to those reported for the purified beta-galactosidase II protein from tomato fruit. J Mol Biol, 1998 May 29, 279(1), 245 - 55 The three-dimensional structure of the nitrogen regulatory protein IIANtr from Escherichia coli; Bordo D et al.; The bacterial rpoN operon codes for sigma 54, which is the key sigma factor that, under nitrogen starvation conditions, activates the transcription of genes needed to assimilate ammonia and glutamate . The rpoN operon contains several other open reading frames that are cotranscribed with sigma 54 . The product of one of these, the 17.9 kDa protein IIANtr, is homologous to IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system . IIANtr influences the transcription of sigma 54-dependent genes through an unknown mechanism and may thereby provide a regulatory link between carbon and nitrogen metabolism . Here we describe the 2.35 A X-ray structure of Escherichia coli IIANtr . It is the first structure of a IIA enzyme from the fructose-mannitol family of the PTS . The enzyme displays a novel fold characterized by a central mixed parallel/anti-parallel beta-sheet surrounded by six alpha-helices . The active site His73 is situated in a shallow depression on the protein surface. Vet Rec, 1998 May 16, 142(20), 545 - 7 Assessing abattoir hygiene with a marker organism; Hudson WR et al.; A study was made to evaluate the use of a marker organism for assessing whether hygienic slaughter practices were being followed at red meat abattoirs . The organism, a nonpathogenic strain of Escherichia coli K12 that was resistant to nalidixic acid, was detected and counted on a highly specific isolation medium . With beef carcases, the practice of bagging the excised anus reduced, but did not prevent the spread of the organism from an inoculum applied in the anal region before the hide was removed . The carcases of sheep that were processed at a low-throughput abattoir, were contaminated with the marker after the fleece had been inoculated at a single site . The contamination was significantly reduced (P<0.001) when the operative responsible for flaying had cleaned his hands, arms and apron before and during the handling of each carcase, and used a knife which was freshly pasteurised on several occasions . However, the subsequent washing of carcases had little or no effect on the levels of the marker organism . It was concluded that the marker may be of value in assessing hygiene control, improving present practices, and training abattoir staff. Int J Immunopharmacol, 1997 Sep-Oct, 19(9-10), 573 - 7 Intervention in autoimmune diabetes by targeting the gut immune system; Bellmann K et al.; BB rats and nonobese diabetic (NOD) mice spontaneously develop autoimmune insulin dependent diabetes and serve as models for human type I diabetes . During progression of the disease the cytokine pattern elaborated by islet infiltrating immune cells shifts from a Th2 or Th0 toward Th1 type . Only the latter is associated with "destructive" insulitis . We discuss here attempts to modulate disease progression by targeting the gut immune system with bacterial immunostimulants . Oral dosing of diabetes prone BB rats with lipopolysaccharide (LPS) or the Escherichia coli extract OM-89 lead to a Th2-shift of pancreatic mRNA expression . In vitro studies showed that repeated exposure toward LPS or OM-89 lead to downregulation of proinflammatory macrophage responses . In the NOD mouse, repeated oral dosing of OM-89 caused a Th2 shift in the gut cytokine gene expression, probably because of desensitization of macrophages and other antigen presenting cells . Concomitantly, diabetes prevention by oral insulin was improved . In conclusion, oral dosing with bacterial immunostimulants dampens Th1 type immune reactivities of the gut immune system and thereby promotes oral tolerance mechanisms . Downregulation of proinflammatory immune reactivities by repeated exposure to bacterial stimulants requires intact desensitization mechanisms in macrophages or other antigen presenting cells. Int J Immunopharmacol, 1997 Sep-Oct, 19(9-10), 569 - 72 Oral dosing of rats with OM-89 results in the appearance of specific OM-89 antibodies of the IgG2a isotype: possible significance in the treatment of rheumatoid arthritis; Phipps PA et al.; OM-89 is a glycoprotein-rich extract of Escherichia coli shown to be effective in the treatment of rheumatoid arthritis (RA) . It has been reported that oral dosing of animals results in the appearance of specific OM-89 antibodies . In the current study we have investigated some of the immunoglobulin isotypes that may be involved . OM-89 antibodies of IgG1, IgG2a and IgM isotypes were measured by ELISA in serum from rats dosed three times a week for 3 weeks at 4 or 40 mg kg(-1) . The results showed a small but significant rise in IgM and a greater rise in IgG2a . The possibility that antigens within OM-89 (e.g . hsp65) may have homology with antigens involved in RA raises the possibility that OM-89 antibodies, particularly of the IgG2 class, may block pathogenic antigens from being recognized by T cells. Int J Immunopharmacol, 1997 Sep-Oct, 19(9-10), 541 - 5 The immunomodulator OM-89 induces ACTH and glucocorticoid secretion in rats through an IL-1 dependent pathway; Mimouni J et al.; The immunomodulator OM-89 (bacterial extract from E . coli), known to act on the immune system by modulating both humoral and cellular responses, significantly increases ACTH and glucocorticoids secretion in normal Wistar rats . A comparative administration of IL-1 displays a similar pattern of release . Moreover, OM-89-induced responses are blocked by IL-1ra, the soluble receptor antagonist of IL-1 . The effect of OM-89 can thus be explained, at least in part, via IL-1 which directly enhances the secretion of both stress hormones . Finally, a comparative experiment between control and stressed rats (by immobilization) shows that the responses to OM-89 measured in this study (ACTH and corticosterone secretion) are stress-modulated. J Vet Med Sci, 1998 May, 60(5), 555 - 61 Dual infection with enterotoxigenic Escherichia coli and porcine reproductive and respiratory syndrome virus observed in weaning pigs that died suddenly; Nakamine M et al.; Diarrhea, sudden death after short duration of diarrhea and sudden death without apparent signs were observed in a herd of breeder pigs . Five pigs that died suddenly with diarrhea (SDD pigs) and 6 pigs that died suddenly without signs (SD pigs) were examined . The average age of the pigs was about 28 days . Twelve pigs of age 10 to 14 days old showing diarrhea (D pigs) were also examined . Eleven of them recovered . Large numbers of Escherichia coli were detected in all organs of every SDD and SD pig and in feces of D pigs . All of the isolates were identified as enterotoxigenic E . coli (ETEC) by the polymerase chain reaction (PCR) . Porcine reproductive and respiratory syndrome (PRRS) virus cDNA was also detected from the lung of every SD and SDD pig by the RT-PCR . High and low titers of antibodies to PRRS virus were found in 10-day-old and 1-month-old pigs, respectively . In an experiment, 3 ETEC were isolated from 9 healthy weaning pigs during the quiescent stage in the herd . These data showed that growth of the ETEC was not active in healthy weaning pigs; however, following infection with PRRS virus ETEC infection became systemic and caused peracute death in the weaning pigs . It suggested also that infection with PRRS virus in 10-day-old pigs were protected by the colostral antibodies, and fatal infection by ETEC did not occur as a result. FEBS Lett, 1998 May 15, 427(3), 377 - 80 Identification of lysine-238 of Escherichia coli biotin carboxylase as an ATP-binding residue; Kazuta Y et al.; Escherichia coli biotin carboxylase was affinity labeled with adenosine diphosphopyridoxal to identify its ATP binding site . Lysyl endopeptidase digestion of the modified protein, followed by high performance liquid chromatography separation and amino acid sequencing allowed to identify lysine-238 to be the site of modification . Site-directed mutagenesis of this residue into alanine, arginine or glutamine resulted in mutants with much decreased activity . Lysine-238 seems to interact with the gamma-phosphate group of ATP but is not involved in catalysis. Mutat Res, 1998 Mar, 407(2), 169 - 76 Activity of Escherichia coli DNA-glycosylases on DNA damaged by methylating and ethylating agents and influence of 3-substituted adenine derivatives; Tudek B et al.; Methylating and ethylating agents are used in the chemical industry and produced during tobacco smoking . They generate DNA base damage whose role in cancer induction has been documented . Alkylated bases are repaired by the base excision repair pathway . We have established the repair efficiency of methylated and ethylated bases by various Escherichia coli repair proteins, namely 3-methyladenine-DNA-glycosylase I (TagA protein), which excises 3-methyladenine and 3-methylguanine, 3-methyladenine-DNA-glycosylase II (AlkA protein), which has a broad substrate specificity including 3- and 7-alkylated purines and the formamidopyrimidine(Fapy)-DNA-glycosylase (Fpg protein) repairing imidazole ring-opened 7-methylguanine . The comparison of the Km values of these various enzymes showed that methylated bases were excised more efficiently than ethylated bases . Several 3-alkyladenine derivatives have been synthesized and examined for their ability to inhibit the activity of the various repair proteins . We have shown that 3-ethyl-, 3-propyl-, 3-butyl- and 3-benzyladenine were much more efficient inhibitors of TagA protein than 3-methyladenine . The inhibitory effect was increased with the increase of the size of alkyl-group and IC50 for 3-benzyladenine was 0.4 +/- 0.1 microM as compared to 1.5 +/- 0.3 mM for 3-methyladenine . These compounds inhibited neither the AlkA protein nor human 3-methyladenine-DNA-glycosylase (ANPG protein) . Moreover, 3-hydroxyethyladenine did not affect the activity of any of these enzymes . Taken together, these results suggest that hydrophobic interactions are involved in the mechanism of inhibition and/or recognition and excision of alkylated purines by TagA protein. Mutat Res, 1998 Mar, 407(2), 117 - 24 A novel plasmid shuttle vector for the detection and analysis of microsatellite instability in cell lines; Diem C et al.; Microsatellite instability is an important feature of tumors from hereditary nonpolyposis colorectal carcinoma (HNPCC) patients as well as a variety of sporadic tumors . Here, we present a novel plasmid shuttle vector for the detection of this replication error (RER+) phenotype in human cell lines . The episomely replicated plasmid pZCA29 harbours the bacterial beta-galactosidase gene interrupted by two palindromically arranged poly-(CA)-repeat tracts . The resulting + 1-frameshift leads to white colonies of Escherichia coli DH10B on X-Gal/IPTG1 agar plates . Mutations in the repeats characteristic of the RER+-phenotype may result in the loss or gain of CA-repeats leading to blue bacterial colonies . We transiently transfected the colorectal cancer cell lines SW480 and HCT116 with the plasmid pZCA29, isolated replicated plasmid DNA after several days and used it to transform E . coli DH10B . We found 1.0 to 1.7% blue colonies after passage of the plasmid through the RER+-cell line SW480 in contrast to 3.5 to 8.1% blue colonies after transfection of the RER+-cell line HCT116, the mutation frequencies increasing with incubation time . Sequence analysis of mutated plasmids revealed mostly 2-bp deletions which occurred especially in one of the repeat tracts . We conclude that pZCA29 appears to be a suitable shuttle vector for the detection and analysis of a RER+-phenotype in cell lines. Mutat Res, 1998 Mar, 407(2), 109 - 16 Repair in Escherichia coli alkB mutants of abasic sites and 3-methyladenine residues in DNA; Dinglay S et al.; Escherichia coli alkB mutants are sensitive to methyl methanesulfonate and dimethylsulphate, and are defective in the processing of methylated DNA . The function of the AlkB protein has not been determined . Here, we show that alkB mutants are not defective in repairing several different types of potentially toxic DNA lesions that are known to be generated by MMS, including apyrimidinic and apurinic sites, and secondary lesions that could arise at these sites (DNA-protein cross-links and DNA interstrand cross-links) . Also, alkB mutants were not sensitive to MeOSO2-(CH2)2-Lex, a compound that alkylates the minor groove of DNA generating primarily 3-methyladenine. Planta, 1998 Jun, 205(2), 263 - 8 The role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids; Schutt BS et al.; To investigate the role of acyl carrier protein (ACP) in determining the fate of the acyl moieties linked to it in the course of de-novo fatty acid biosynthesis in higher plants, we carried out in vitro experiments to reconstitute the fatty acid synthase (FAS) reaction in extracts of spinach (Spinacia oleracea L.) leaves, rape (Brassica napus L.) seeds and Cuphea lanceolata Ait . seeds . The action of two major C . lanceolata ACP isoforms (ACP 1 and ACP 2) compared to ACP from Escherichia coli was monitored by saponification of the corresponding FAS products with subsequent analysis of the liberated fatty acids by high-performance liquid chromatography . In a second approach the preference of the medium-chain acyl-ACP-specific thioesterase (EC 3.1.2.14) of C . lanceolata seeds for the hydrolysis of acyl-ACPs prepared from the three ACP types was investigated . Both ACP isoforms from C . lanceolata seeds supported the synthesis of medium-chain fatty acids in a reconstituted FAS reaction of spinach leaf extracts . Compared to the isoform ACP 1, ACP 2 was more effective in supporting the synthesis of such fatty acids in the FAS reaction of rape seed extracts and caused a higher accumulation of FAS products in all experiments . No preference of the medium-chain thioesterase for one specific ACP isoform was observed . The results indicate that the presence of ACP 2 is essential for the synthesis of decanoic acid in C . lanceolata seeds, and its expression in the phase of accumulation of high levels of this fatty acid provides an additional and highly efficient cofactor for stimulating the FAS reaction. J Mol Biol, 1998 May 29, 279(1), 189 - 99 Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites; Steinmetz MO et al.; Recombinant rat liver GTP cyclohydrolase I has been prepared by heterologous gene expression in Escherichia coli and characterized by biochemical and biophysical methods . Correlation averaged electron micrograph images of preferentially oriented enzyme particles revealed a fivefold rotational symmetry of the doughnut-shaped views with an average particle diameter of 10 nm . Analytical ultracentrifugation and quantitative scanning transmission electron microscopy yielded average molecular masses of 270 kDa and 275 kDa, respectively . Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homodecameric protein complex consisting of two fivefold symmetric pentameric rings associated face-to-face . Examination of the amino acid sequence combined with calcium-binding experiments and mutational analysis revealed a high-affinity, EF-hand-like calcium-binding loop motif in eukaryotic enzyme species, which is absent in bacteria . Intrinsic fluorescence measurements yielded an approximate dissociation constant of 10 nM for calcium and no significant binding of magnesium . Interestingly, a loss of calcium-binding capacity observed for two rationally designed mutations within the presumed calcium-binding loop of the rat GTP cyclohydrolase I yielded a 45% decrease in enzyme activity . This finding suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I gene of patients suffering from dopa responsive dystonia. J Mol Biol, 1998 May 29, 279(1), 31 - 48 Mutations affecting cooperative DNA binding of phage HK022 CI repressor; Mao C et al.; Cooperative protein-DNA interactions play critical roles in gene regulation in all organisms . Among the best-studied cooperative interactions is that of phage lambda repressor, which binds cooperatively to two adjacent operators . Similar cooperative interactions are also shown by several other lambdoid phage repressors, including HK022 CI repressor, which we study here . This protein has a much higher degree of cooperativity than seen with lambda repressor, and previous evidence has suggested that cooperativity may play roles in HK022 gene regulation that have no parallel in lambda . We have isolated several cooperativity or Coop- mutations in HK022 cI . These mutant proteins were partially defective in vivo for binding to two adjacent operators, but normal or nearly so for binding to a single operator . Two mutations showed mutual suppression, in that the double mutation had wild-type behavior . Analysis of several purified mutant proteins showed that they were also defective for cooperative binding in vitro . Unexpectedly, the mutant proteins showed an altered pattern of in vitro binding to DNA at non-operator sites . Several of them also increased the rate of specific repressor cleavage . We propose a conformational model in which the various functions of the wild-type protein are carried out by differing conformations; these conformations are normally in balance, and the mutations perturb this balance. Virology, 1998 Jun 5, 245(2), 344 - 52 Identification of two replicons in phage-plasmid P4; Tocchetti A et al.; DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr . Replication depends on the product of the P4 alpha gene, a protein with primase and helicase activity, that binds both ori1 and crr . A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4 . Using a plasmid complementation test for replication, we found that two replicons, both dependent on the alpha gene product, coexist in P4 . The first replicon is made by the cnr and alpha genes and the ori1 and crr sites . The second is limited to the alpha and crr region . Thus, in the absence of the ori1 region, replication can initiate at a different site . By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene . The ori2 site was found to be dispensable in a replicon that contains ori1 . A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2. Biotechnology (N Y), 1996 Jan, 14(1), 61 - 5 From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis; Wilkins MR et al.; Separation and identification of proteins by two-dimensional (2-D) electrophoresis can be used for protein-based gene expression analysis . In this report single protein spots, from polyvinylidene difluoride blots of micropreparative E . coli 2-D gels, were rapidly and economically identified by matching their amino acid composition, estimated pI and molecular weight against all E . coli entries in the SWISS-PROT database . Thirty proteins from an E . coli 2-D map were analyzed and identities assigned . Three of the proteins were unknown . By protein sequencing analysis, 20 of the 27 proteins were correctly identified . Importantly, correct identifications showed unambiguous "correct" score patterns . While incorrect protein identifications also showed distinctive score patterns, indicating that protein must be identified by other means . These techniques allow large-scale screening of the protein complement of simple organisms, or tissues in normal and disease states . The computer program described here is accessible via the World Wide Web at URL address . Biotechnology (N Y), 1996 Jan, 14(1), 50 - 5 Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging; Thomann HU et al.; Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies . Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination . However, as it is not possible to express noncomplementing mutant enzymes in an E . coli glnS-deletion strain, we developed a novel strategy to address these problems . Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E . coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts . Here we report the use of E . coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity . In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods . In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope . The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging. Biotechnology (N Y), 1995 Sep, 13(9), 988 - 93 A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide; Dobeli H et al.; Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta) . A 42-residue peptide (A beta 1-42) appears to be the predominant form . In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate . A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production . The method is based on a protein tail fused to the amino terminus of A beta . This tail leads to a high expression in E . coli, and a histidine affinity tag facilitates purification . Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column . Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta . Furthermore, immobilization prevents aggregation of cleaved A beta . Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer . Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours . This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities . Mutations of A beta 1-42 at position 35 influence the aggregation properties . Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue . The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue. Biotechnology (N Y), 1995 Sep, 13(9), 982 - 7 Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA; Collins-Racie LA et al.; Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum . The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys . This stringent site specificity gives EK great potential as a fusion protein cleavage reagent . Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells . We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E . coli DsbA protein as an N-terminal fusion partner . The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site . Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction . The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme . Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena . Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence. Biotechnology (N Y), 1995 Sep, 13(9), 974 - 81 Design, expression, and initial characterization of MB1, a de novo protein enriched in essential amino acids; Beauregard M et al.; Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine . Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure . We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold . The design process was an iterative one . The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini . Using these data, the helices in our de novo protein were designed by inserting the residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices . Short sequences of strong turn formers were used to join the helices and adjust the predicted p1 to 7.7, while a number of local and global factors were used to refine our design . Further, the sequence was checked to eliminate various known protease targets in E . coli . The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNKE DMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA+ ++ , where dashes separate long helices from short, turn forming linkers . A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter . The fusion protein was expressed in E . coli, purified and cleaved to yield maltose binding protein and our de novo protein, MB1 . MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L. Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7463 - 8 A chloroplast processing enzyme functions as the general stromal processing peptidase; Richter S et al.; A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide . The identity of this key component of the import machinery has not been unequivocally established . We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) . Here we report the overexpression of active CPE in Escherichia coli . Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle . CPE also processes its own precursor in trans . Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate . The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well . These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase . Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc. Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7391 - 5 Mass spectrometry of ribosomes and ribosomal subunits; Benjamin DR et al.; Nanoflow electrospray ionization has been used to introduce intact Escherichia coli ribosomes into the ion source of a mass spectrometer . Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer . Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins . The pattern of dissociation correlates strongly with predicted features of ribosomal protein-protein and protein-RNA interactions . The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome . This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.
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