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<